Integrin targeted synthetic ligands for diagnostic and therapeutic applications

ABSTRACT

The present invention relates to a novel class of diagnostically or therapeutically effective compounds comprising novel aza-bicycloalkane based cyclic peptides, acting as a targeting moiety towards integrin receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/885,353,filed Nov. 23, 2007, allowed; which is the national stage of ApplicationNo. PCT/IB2006/000455, filed Mar. 3, 2006; which claims priority benefitof Italian Application No. MI2005A000328, filed Mar. 3, 2005; the entirecontents of which are incorporated by reference.

TECHNICAL FIELD

The present invention is in the technical field of the targeteddiagnostic imaging and relates to novel cyclic peptidomimetic compoundshaving an azabicycloalkane structure conjugated to a biologically activemolecule.

In particular, the invention relates to a novel class of diagnosticallyor therapeutically effective compounds comprising novelaza-bicycloalkane based cyclic peptides, acting as a targeting moietytowards integrin receptors.

The invention further relates to novel pharmaceutical compositionscomprising them and uses thereof in targeted imaging or therapy of solidtumors and in general, of the pathological conditions associated withangiogenesis.

BACKGROUND OF THE INVENTION

A great number of physiological processes involve biologically activepeptides, through their interactions with receptors and enzymes.However, peptides are not to be considered ideal drugs, given their poormetabolic stability, rapid excretion and low selectivity for specificreceptors. A valid alternative involves the design of peptide analogueswhich are capable of mimicking the action of the natural peptide at thereceptor level (peptidomimetic) [(a) Kahn, M. (Editor). PeptideSecondary Structure Mimetics. Tetrahedron Symposia-in-Print No. 50 1993,49, 3433-3689. (b) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33,1699-1720. (c) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bös, M.; Cook,C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; Kahn, M.;Madison, V. S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall, J.; Vincent, G.P.; Voss, M. E. J. Med. Chem. 1993, 36, 3039-3049. (d) Kitagawa, O.;Velde, D. V.; Dutta, D.; Morton, M.; Takusagawa, F.; Aubé, J. J. Am.Chem. Soc. 1995, 117, 5169-5178. (e) Giannis, A.; Kolter, T. Angew.Chem.; Int. Ed. Engl. 1993, 32, 1244. (f) Aube, J. TetrahedronSymposia-in-Print No. 50, 2000, 56, 9725-9842].

During our research into peptide secondary structure mimetics, certain6,5- and 7,5-azabicycloalkane aminoacids have been synthesised [(a)Colombo, L.; Di Giacomo, M.; Scolastico, C.; Manzoni, L.; Belvisi, L.;Molteni, V. Tetrahedron Lett. 1995, 36, 625; (b) Colombo, L.; DiGiacomo, M.; Belvisi, L.; Manzoni, L.; Scolastico, C. Gazz. Chim. It.1996, 126, 543; (c) Colombo, L.; Di Giacomo, M.; Brusotti, G.; Sardone,N.; Angiolini, M.; Belvisi, L.; Maffioli, S.; Manzoni, L.; Scolastico,C. Tetrahedron 1998, 54, 5325-5336; (d) Angiolini, M.; Araneo, S.;Belvisi, L.; Cesarotti, E.; Checchia, A.; Crippa, L.; Manzoni, L.;Scolastico, C. Eur. J. Org. Chem. 2000, 2571-2581; (e) Manzoni, L.;Colombo, M.; May, E.; Scolastico, C. Tetrahedron 2001, 57, 249; (f)Belvisi, L.; Colombo, L.; Colombo, M.; Di Giacomo, M.; Manzoni, L.;Vodopivec, B.; Scolastico, C. Tetrahedron 2001, 57, 6463; (g) EP 1 077218; (h) Colombo, L.; Di Giacomo, M.; Vinci, V.; Colombo, M.; Manzoni,L.; Scolastico, C. Tetrahedron, 2003, 59, 4501-4513; (i) Manzoni, L.;Colombo, M.; Scolastico, C. Tetrahedron Lett. 2004, 45, 2623-2625; (l)Belvisi, L.; Colombo, L.; Manzoni, L.; Potenza, D.; Scolastico, C.Synlett, 2004, 1449-1471.

These structures may be considered as conformationally constrainedanalogues of the Ala-Pro and Phe-Pro dipeptide units. [(a) Belvisi, L.;Bernardi, A.; Manzoni, L.; Potenza, D.; Scolastico, C. Eur. J. Org.Chem. 2000, 2563-2569; (b) Gennari, C.; Mielgo, A.; Potenza, D.;Scolastico, C.; Piarulli, U.; Manzoni, L. Eur. J. Org. Chem. 1999, 379].

The functionalisation of such molecules with heteroalkyl substituents isan aim of great interest, since the side chains may increase theaffinity of the peptide for the receptor by interacting with thehydrophobic or hydrophilic sites of the receptor itself. A furtheradvantage of such systems is the possibility of binding to differentpharmacophoric groups and hence the possibility of creating a library,with the member components of which having different biologicalproperties and activities. During our research into peptide secondarystructure mimetics, certain 6,5- and 7,5-azabicycloalkane aminoacidshave been synthesised which have been functionalised with heteroalkylappendages [(a) Artale, E.; Banfi, G.; Belvisi, L.; Colombo, L.;Colombo, M.; Manzoni, L.; Scolastico, C. Tetrahedron, 2003, 59,6241-6250; (b) Bracci, A.; Manzoni, L.; Scolastico, C. Synthesis 2003,2363-2367; (c) Bravin, F. M.; Busnelli, G.; Colombo, M.; Gatti, F.;Manzoni, L.; Scolastico, C. Synthesis, 2004, 353; (d) Manzoni, L.;Belvisi, L.; Colombo, M.; Di Carlo, E.; Formi, A.; Scolastico, C.Tetrahedron Lett. 2004, 45, 6311-6315].

Furthermore, analogously to what occurs for non-substitutedconformationally constrained dipeptide mimetics [Belvisi, L.; Bernardi,A.; Checchia, A.; Manzoni, L.; Potenza, D.; Scolastico, C.; Castorina,M.; Cupelli, A.; Giannini, G.; Carminati, P.; Pisano, C. Org. Lett.2001, 3, 1001, C. Scolastico, L. Manzoni, G. Giannini. Brit. UK Pat.Appl. 2004. GB 2395480] such heteroalkyl substituted lactams may beincorporated into cyclic pseudo-peptides containing RGD sequence.

Such molecules may be selectively targeted to those tissuesover-expressing certain receptors (e.g. epithelial cells involved invascular growth), so as to be able to be used to inhibit angiogenesisand selectively control the release of any drugs optionally bound to thesubstituent groups on the lactam ring [Arap, W.; Pasqualini, R.;Ruoslahti, E. Science, 1998, 279, 377]. Thus, from a first point ofview, the low number of “scaffolds” reported in the literaturenecessitates the design and synthesis of novel conformationallyconstrained dipeptide mimetics, functionalised with hetero-substitutedside chains for interaction with various receptors.

Moreover, and from another relevant point of view, today is well knownthat the cell adhesion molecule α_(v)β₃ is an important player in theprocess of tumor angiogenesis and metastasis. With the term angiogenesisis identified a formation process of new blood vessels that occurs notonly during embryonic development and normal tissue growth and repair,but is also associated with the female reproductive cycle, establishmentand maintenance of pregnancy, and repair of wounds and fractures. Inaddition to angiogenesis that occurs in the normal individual,angiogenic events are involved in a number of pathological processes,notably tumor growth and metastasis, and other conditions in which bloodvessel proliferation is increased, such as diabetic retinopathy,psoriasis and arthropathies. Angiogenesis is so important in thetransition of a tumor from hyperplastic to neoplastic growth, thatinhibition of angiogenesis has become an active cancer therapy researchfield. Tumor angiogenesis differs significantly from physiologicalangiogenesis. The differences include aberrant vascular structure,altered endothelial cell-pericyte interactions, abnormal blood flow,increased permeability and delayed maturation. Molecules regulatingangiogenesis include growth factor receptors, tyrosine kinase receptors,G protein-coupled receptors for angiogenesis modulating proteins, andintegrins [Bergers G., L. E. Benjamin, Nat. Rev. Cancer, 2003,3:401-410; Ferrara N., Nat. Rev. Cancer, 2002, 2:795-803, Nyberg, P., L.Xie, R. Kalluri, Cancer Res., 2005, 65:3967-3979]. Increasing amounts ofevidences now imply that integrin signaling plays a key role in tumorangiogenesis and metastasis [Brooks, P. C., R. A. Clark, D. A. Cheresh,1994, Science, 264:569-571, Kumar, C. C., 2003, Curr. Drug Targets,4:123-131]. The α_(v)β₃ integrin, in particular, is significantlyup-regulated on endothelium during angiogenesis but not on quiescentendothelium [Hood, J. D. and D. A. Cheresh, 2002, Nat. Rev. Cancer,2:91-1000, Xiong, J. P., T. Stehle, R. Zhang, A. Joachimiak, et al.,2002, Science, 296:151-155, Jin, H. and J. Varner, 2004, Br. J. Cancer,90:561-565]. Research has shown that tumor expression of integrinα_(v)β₃ correlates well with tumor in several malignancy such asmelanoma, glioma, ovarian cancer, and breast cancer. Thus, the abilityto quantitatively image integrin α_(v)β₃ expression in vivo in anon-invasive manner may shed new light into the mechanism ofangiogenesis and antiangiogenic treatment efficacy based on integrinantagonism. Tumor integrin expression imaging will also aid in lesiondetection, to more appropriately select patient for anti-integrintreatment, in new anti-integrin drug development/validation, as well asin treatment monitoring and optimization.

Kessler and coworkers have provided new cyclic RGD based pentapeptidesincluding the optimized c(RGDfV) system [see, for instance,US2002/0198142 and EP 0632053, U.S. Pat. No. 6,001,961] wherein the saidpentapeptides are proposed for use, as such, as integrin-inhibitor drugsin the control of diseases, in particular disorders of the circulation,thrombosis, cardiac infarction, arteriosclerosis, inflammations, andangiogenesis. Cited references, however, do not suggest the use ofclaimed peptides or of suitable derivatives thereof for the diagnosticimaging or radiotherapy of angiogenesis and angiogenic disorders.

EP1068244 discloses pharmaceuticals useful for the diagnosis andtreatment of cancer comprising peptides or peptidomimetics targeted toreceptors up-regulated during angiogenesis and chelators.

Diagnostic or therapeutic agents including the RGD sequence and,particularly, the above c(RGDfV) cyclic peptides targeted to integrinreceptors are also known: [Janssen, M. L., W. J. Oyen, I. Dijkgraaf, etal. 2002, Cancer Res. 62:6146-6151; Haubner, R. H., H. J. Wester, et al.Cancer Res. 61:1781-1785; Haubner, R. H., H. J. Wester, et al. 2003, Q.J. Nucl. Med. 47:189-199; Haubner, R. H., H. J. Wester, 2004, Curr.Pharm. Des. 10:1439-1455; Wang, W., S. Ke, et al. 2004, Mol. Imaging3:343-351; Sunkuku, K., K. Shi, et al., 2005, Mol Imaging, 4:75-87,Sipkins D. A., D. A. Cheresh, et. al., Nat. Med. 4:623-626].

The said prior art agents, however, generally suffer of drawbacksderiving from poor diffusion or transport, limited availability, and/orlack of specificity all resulting in a modest tumor-to-background ratio,and very low contrast observed in angiogenic regions.

Thus still remains a need for diagnostic and therapeutic agents that,when administered in vivo to a mammal, may combine high specificity andacceptable pharmacokinetic properties.

SUMMARY OF THE INVENTION

The present invention relates to novel therapeutic or diagnostic agentscomprising cyclic peptide derivatives able to selectively bind tointegrin receptors.

In particular, the cyclic peptide derivatives of the invention comprisean aza-bicycloalkane structure, acting as conformationally constrainedmimetic of the omoSer-Pro dipeptide, that is further conjugated to abiologically active molecule. The said peptide derivatives are able toselectively bind to integrin receptors thus they act, according to thepresent invention, as targeting moiety able to bring and successfullybound an active moiety linked thereto to this particular class ofreceptors.

This class of diagnostic or therapeutic agents may find application forthe diagnosis, prevention and treatment of pathological conditionsassociated with angiogenesis.

As detailed in the following paragraphs, the present invention relatesto a novel class of compounds; to a manufacturing process for theirpreparation; to these same compounds for use as therapeutic ordiagnostic agents; to the use of these latter in the preparation ofpharmaceutical compositions for treatment, prevention and imaging ofangiogenesis and of pathological conditions associated thereto.

According to a still different embodiment of the invention, alsoprovided is a method for the treatment and prevention of angiogenesisand of related disorders, as well as a method for imaging angiogenesisboth in vitro and in vivo, the said methods comprising theadministration and use of the compounds of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 comprise synthetic schemes detailing the preparation of thepeptidomimetic compounds of the invention. In particular:

FIG. 1 discloses, schematized in Scheme 1, the synthetic process for thepreparation of compounds of general formulae (Ia) 6,5-trans- and (Ib)6,5-cis-fused, wherein n=1;

FIG. 2 discloses, schematized in Scheme 2, the synthetic process for thepreparation of compounds of general formulae (Ia) 7,5-trans and (Ib)7,5-cis fused, wherein n=2;

FIG. 3 discloses, schematized in Scheme 3, the cyclization process.

FIGS. 4-5 disclose, schematized in Scheme 4 and 5 the preparation of thebiologically active derivatives of the invention;

(IIa) and (IIb) and of a biologically active derivative thereof; inparticular in FIG. 4, scheme 4, is reported the preparation ofderivative 6,5- and 7,5- cis and in FIG. 5, scheme 5, is reported thepreparation of the 6,5- and 7,5-trans derivative.

FIG. 6-Scheme A—discloses the preparation of the starting products.

FIGS. 7A, 7B, and 7C illustrate examples of anchoring systems employedfor preparing multimeric constructs of the invention.

FIGS. 8A, 8B, and 8C illustrate examples of preferred chelators foreither ¹¹¹In and lanthanides such as paramagnetic Gds' or radioactivelanthanides such as, for example, ⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, and ¹⁶⁶HO.

FIGS. 9A and 9B illustrate examples of preferred chelators ofradioactive metal ion such as ^(90m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re.

FIG. 10 reports the ¹H-NMR spectrum (14.1 T, 298 K) of the preparationof the of biotinylated LIPOCEST agent of Example 1. The chemical shiftdifference between intraliposomal and bulk water was 3 ppm.

FIG. 11 reports the normalised Z-spectrum of the of the preparation ofthe LIPOCEST compound of Example 1 (7 T, irrad. pulse: rectangular,irrad. power 6 mT, irrad. time 2 s, 312 K).

FIG. 12 reports the corresponding ST-spectrum of the preparation of theLIPOCEST compound of Example 1. A Saturation Transfer (ST) % of ca. 70%was observed at 3 ppm from bulk water.

FIG. 13 reports the ¹H-NMR spectrum (14.1 T, 298 K) of the preparationof the biotinylated LIPOCEST agent of Example 2.

FIG. 14 reports the normalised Z-spectra of diluited samples of thepreparation of the biotinylated LIPOCEST agent of Example 2 (7 T, irrad.pulse: rectangular, irrad. power 6 mT, irrad. time 2 s, 312 K).

FIG. 15 reports the ¹H-NMR spectrum (14.1 T, 298 K) of the preparationof the LIPOCEST of Example 3. The chemical shift difference betweenintraliposomal and bulk water was 10 ppm.

FIG. 16 reports the ¹H-NMR spectrum (14.1 T, 298 K) of the of thepreparation of the LIPOCEST of Example 4. The chemical shift differencebetween intraliposomal and bulk water was 19.3 ppm.

FIG. 17 reports the ¹H-NMR spectrum (14.1 T, 298 K) of the preparationof the LIPOCEST of Example 5. The chemical shift difference betweenintraliposomal and bulk water was −36 ppm.

FIG. 18 reports the ¹H-NMR spectrum (14.1 T, 298 K) of the preparationof the LIPOCEST of Example 6. The chemical shift difference betweenintraliposomal and bulk water was 3 ppm.

FIG. 19 reports the ST spectra of the two cellular pellets of Example 7,including, the first HUVEC cells treated with the biotinilated LIPOCESTand the second only HUVEC cells as control (7 T, irrad. pulse:rectangular, irrad. power 12 μT, irrad. time 2 s, 293 K).

FIG. 20 reports the CEST-MR images registered for the two cellularpellets of example 7, [on-(irradiation at 10 ppm from bulk water),off-(irradiation at −10 ppm from bulk water), and (on-off)] of thephantom made of two capillaries containing two cellular pellets.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel therapeutic or diagnostic agentscomprising cyclic peptide derivatives able to selectively bind tointegrin receptors. In particular, it is a first object of the inventiona compound of formula (III)

whereinn is 1 or 2,p is an integer between 1 and 5,R₄ and R₅ together constitute the sequence Asp-Gly-Arg,R₆ is a biologically active moiety,L is a group (i) —CONH—, (ii) —NHCONH—, (iii) —NHCSNH—, a group offormula

or it is a divalent linking moiety connecting R₆ to the —(CH₂)p-bicycloderivative in formula (III), through any one of the above (i) to (v)groups, their salts, racemic mixtures, individual enantiomers,individual diastereoisomers and mixtures thereof in whatever proportion.

Within the compounds of formula (III), the peptide sequence Asp-Gly-Argis advantageously bound to compounds in such a manner whereby thecarboxyl group is attached to the aminoacid arginine, and the aminogroup is attached to aspartic acid.

According to preferred embodiment of the invention, p is 1 and n is 1.

According to another preferred embodiment of the invention, p is 1 and nis 2.

According to another preferred embodiment of the invention, within thecompounds of formula (III), L is a group from (i) to (v).

As formerly reported, the compounds of the invention are able toselectively bind to integrin receptors, in particular αvβ3 and αvβ5integrins overexpressed into angiogenic tissues. These compounds maythus optimally bind a cell surface or an intravascular surfaceexpressing the targeted receptor wherein this results in an optimalselectivity and localization of the biological activity they mayexpress.

Thus, this class of compounds may find application for the diagnosis,prevention and treatment of pathological conditions associated withangiogenesis.

In the present description, unless otherwise provided, with the term“Angiogenesis” as used herein we intend an invasive multi-step processcharacterized by endothelial cell proliferation, modulation ofextarcellular matrix (ECM), and cell adhesion and migration, resultingin the formation of new blood vessels and/or in the increase in thevascularity of an organ or tissue of the body.

With the term “angiogenic events or processes” as used herein we intendthe events or processes involved in a number of pathological conditions,notably tumor growth and metastasis, and other conditions in which bloodvessel proliferation is increased such as diabetic retinopathy,psoriasis and arthropathies.

The term “angiogenic disorders”, angiogenic diseases” and “pathologicalconditions associated with angiogenesis” are used herein interchangeablyto referrer to clinical conditions involving up-regulation of anangiogenic process leading to excessive blood vessel formation. Theseconditions include, but are not limited to, cancer, psoriasis,atherosclerosis, restenosis, a number of inflammatory disorders such as,for example, rheumatoid arthritis, and ocular neovascularizationleading, in most cases to diabetic retinopathies, neovascular glaucoma,age-related macular degeneration or retinal vein occlusion.

The term “integrin”, as used herein, refers to any of the many cellsurface receptor proteins, also known as adhesion protein receptors,which bind extracellular matrix ligand or other cell adhesion proteinligands and thereby mediate cell-cell and cell-matrix adhesionprocesses. Integrins constitute a superfamily of membrane receptors thatare encoded by genes belonging to a gene superfamily and are typicallycomposed of heterodynamic transmembrane glycoproteins containing an α-and a β-subunit. Members of an integrin subfamily have a common βsubunit, which can combine with different α subunits to form adhesionprotein receptors with different specificities. Among the known αsubunits, the α_(v) subunit seems to be the most promiscuous, formingheterodimers with six different β subunits. Integrin α_(v) include, forexample, the receptors α_(v)β₃ and α_(v)β₅.

With the term “integrin binding moiety”, as used herein, we intend anintegrin-inhibiting moiety that specifically acts by binding tointegrins, thereby precluding, reversing, inhibiting or otherwiseinterfering with the binding of integrins to their endogenous ligands.Preferably, integrin binding moieties exhibit a high binding affinityand specificity for α_(v) integrins; more preferably, for the integrinsα_(v)β₃ and/or α_(v)β₅; most preferably for the α_(v)β₃ integrin. Whenan integrin binding moiety is part of a molecule, it confers itsproperty to the molecule, and the said molecule become “targeted” tointegrins, i.e. this molecule specifically and efficiently binds tointegrins. The binding between integrins and an integrin binding moietymay be covalent or non-covalent, including, in this last case,hydrophobic interactions, electrostatic interactions, Van der Waalsinteractions, hydrogen bonding etc. Most often the binding isnon-covalent.

With the term “integrin antagonist” and “integrin inhibitor” as usedherein interchangeably we intend molecules, agents, or compounds able toinhibiting the biological activity of integrins.

With the term “binding affinity” and “affinity” as used hereininterchangeably, we refer to the level of attraction between molecularentities. Affinity can be expressed quantitatively as a dissociationconstant (K_(d)) or its inverse, the association constant (K_(a)). Inthe context of this invention, as reported below, two types of affinityare considered: (a) the affinity of an integrin binding moiety forintegrins and (b) the affinity of metal chelates moiety for a transitionmetal or another metal entity.

Within the compounds of formula (III), R₆ designates a biologicallyactive moiety.

With the term “biologically active molecule”, as used herein, we intendany therapeutically or diagnostically effective molecule, a sugar, adrug, a phospholipid or lipid moiety, a biotin or an avidin residue thatis linked to the targeting moiety through (L).

According to an aspect of the invention, R₆ represents a therapeuticallyor diagnostically effective molecule.

In a preferred embodiment of the invention R₆ represents an imagingdetectable moiety.

With the term “imaging detectable moiety” and “imaging moiety” as usedherein interchangeably we intend any moiety detectable by imagingprocedures, that is to say any moiety able to provide, to improve or, inany way, to advantageously modify the signal detected by an imagingdiagnostic technique today in use including, for instance, magneticresonance imaging, radioimaging, ultrasound imaging, x-ray imaging,light imaging, thus enabling the registration of diagnostically useful,preferably contrasted, images when used in association with the saidtechniques.

Suitable examples of the said imaging detectable moieties include, forinstance, chelated gamma ray or positron emitting radionuclides;paramagnetic metal ions in the form of chelated or polychelatedcomplexes as well as of micellar systems, liposomes and microspheres;magnetic, diamagnetic or superparamagnetic coated particles,microparticles, nanoparticles; X-ray absorbing agents including atoms ofatomic number higher than 20; bubbles, microbubbles balloons andmicroemulsions including biocompatible gas; a dye molecule; afluorescent molecule; a phosphorescent molecule; a molecule absorbing inthe UV spectrum; a quantum dot; a molecule capable of absorption withinnear or far infrared radiations; optionally coated particles,microparticles and nanoparticles including perfluorocarbons and, ingeneral, all moieties which generate a detectable substance.

A wide range of materials detectable by diagnostic imaging modalities isknown in the art and the imaging modality to be used may be selectedaccording to the imaging detectable moiety the diagnostic compounds ofthe invention include.

With the term “chelator”, “metal chelating ligand” and “chelatingligand” as used herein interchangeably we intend chemical moieties,agents, compounds, or molecules characterized by the presence of polargroups able to a form a complex containing more than one coordinate bondwith a transition metal or another metal entity. In a preferred aspectof the invention the said chelating ligand includes cyclic or linearpolyamino polycarboxylic or phosphonic acids.

With the term “contrast imaging agent” or “contrast agent” as usedherein, we refer to any detectable entity that can be used to in vitroor in vivo visualize or detect a biological element including cells,biological fluids and biological tissues originating from a live mammalpatient, and preferably, human patient, as well as human body organ,regions or tissues affected by angiogenesis when the said detectableentity are used in association with a suitable diagnostic imagingtechnique.

With the term “metal entity” as used herein we intend a paramagneticmetal ion that is detectable by imaging techniques such as MagneticResonance Imaging (MRI), or to a radionuclide that is detectable byimaging techniques such as Single Photon Emission Computed Tomography(SPECT) and Positron Emission Tomography (PET).

MRI Contrast Agents

As formerly reported, and according to a preferred embodiment of theinvention, within the compounds of formula (III) R₆ is an imagingdetectable moiety.

Even more preferably, R₆ represents one or more paramagnetic metal ionin the form of a chelated or polychelated complex, or a suitablephysiologically acceptable salt thereof.

Suitable paramagnetic metal ions for use in the present inventioninclude any of the paramagnetic metal ions known in the art as contrastenhancers in MRI and easily incorporated into chelating or polychelatingmoieties.

Preferably, the paramagnetic metal ion is selected from the following:Fe(²⁺), Fe(³⁺), Cu(²⁺), Ni(²⁺), Rh(²⁺), Co(²⁺), Cr(3+), Gd(³⁺), Eu(³⁺),Dy(³⁺), Tb(³⁺), Pm(³⁺), Nd(³⁺), Tm(³⁺), Ce(³⁺), Y(³⁺), Ho(³⁺), Er(³⁺),La(³⁺), Yb(³⁺), Mn(³⁺), Mn(²⁺). More preferably, the paramagnetic metalion is Gd(³⁺).

A suitable metal chelating moiety may be any metal chelator,polychelator and metal complexing molecule that is able to bind withhigh affinity the said paramagnetic ions so as to provide highly stable,non-toxic, paramagnetic chelated complexes still leaving at least onecoordination site open for a water molecule. A number of either linearor cyclic metal chelating moieties are known in the art including acids,for instance bearing methylene phosphoric acidic groups, methylenehydroxamic acidic groups, carboxyethylidene groups, or carboxymethylenegroups.

Examples of chelators include, but are not limited to,diethylenetriamine pentaacetic acid (DTPA) and derivatide thereofincluding, for example, benzo-DTPA, dibenzo-DTPA, phenyl-DTPA,diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA;1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10 teraazacyclododecanetriacetic acid (DO3A);1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic acid(DOTMA), ethylenediaminetetraacetic acid (EDTA); and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG) and derivatives thereof, including 5-Cl-EHPG, 5-Br-EHPG,5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; bis-2(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivativesthereof; the class of macrocyclic compounds which contain at least 3carbon atoms, more preferably at least 6, and at least two heteroatoms(O and/or N), which macrocyclic compounds can consist of one ring, ortwo or three rings joined together at the hetero ring elements, e.g.,benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM).Preferred chelators and chelates contemplated by the present inventionare further described in WO 2005/062828, WO2003/008390 and EP1155023that are incorporated herein by reference.

Further examples of preferred chelators according to the presentinvention are included in FIGS. 8A to 8C together with suitablebibliographic references concerning their preparation.

Polychelators or polychelating molecules or moieties, according to thepresent invention, include more than 50, preferably more than 100, andeven more preferably more than 300 chelating units, suitably linked to apolymeric chain. Suitable examples of the said polychelators comprise,but are not limited to, suitably functionalized poly-amino acid chainsincluding polylysine, polyalbumine and poly-saccharidic chains includingpolydextranes, dendrimers, and polymeric and copolymeric derivativeslinking up to 300 chelating units. See, for a reference to the saidpolychelating units, WO 91/05762, US5958373, WO 90/12050, WO94/27498,EP0888129, U.S. Pat. No. 600,934, U.S. Pat. No. 5,517,993, WO93/06868,US20050085417, EP1151997, WO9306148, U.S. Pat. No. 6,274,713, WO95/24225, U.S. Pat. No. 5,679,810, EP0607222, WO9507270, EP 0000378,WO9105762, WO 2003/014157, EP 0722442, WO 2003/014157, WO 97/01359, andWO 97/32862.

Examples of preferred MRI contrast agents of the invention comprise

Therapeutically Effective Agents

In a further aspect R₆ is a therapeutic agent or a toxin for selectivekilling and/or inhibiting the growth of cancer cells, or, when required,for inhibiting or promoting the growth of normal tissues.

In a preferred embodiment of the invention, R₆ is a suitably chelatedradioactive metal ion that emits ionizing radiations such as betaparticles, alpha particles and Auger or Coster-Kroning electrons, thusproviding a radiopharmaceutical agent able to target the saidradioactive metal ion to the tumor neovasculature; hence, the beta oralpha-particles emitting radioisotope emits a cytotoxic amount ofionizing radiation causing the cell death.

For therapeutic purposes, the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au.

These pharmaceutical agents of the invention comprising a particleemitting radioactive metal ion are also useful for treating rheumatoidarthritis wherein the growth of a highly vascularized pannus is causedby the excessive production of angiogenic factors by infiltratingmacrophages, immune cells, or inflammatory cells. The locallyconcentrated emission of cytotoxic radiation provided by the therapeuticagents of the invention thus promote the destruction of the newangiogenic vasculature, thus allowing the therapeutic treatment of thisinflammatory process.

Nuclear Imaging (Radionuclide Imaging) and Radiotherapy

According to an alternative embodiment of the invention, R₆ is asuitably chelate gamma ray or positron emitting radionuclide or aradionuclide for therapy.

Compounds of the invention comprising the said R₆ moiety may be used ascontrast agents for scintigraphy, PET or SPECT.

Preferred metal radionuclides for use in PET imaging are positronemitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As, ^(94m)Tc, or¹¹⁰In.

Preferred metal radionuclides for scintigraphy or radiotherapy include^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb,¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, 109Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metalwill be determined based on the desired therapeutic or diagnosticapplication. For example, for diagnostic purposes the preferredradionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In. Fortherapeutic purposes, the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au. ^(99m)Tc is particularly preferred fordiagnostic applications because of its low cost, availability, imagingproperties, and high specific activity. The nuclear and radioactiveproperties of ^(99m)Tc make this isotope an ideal scintigraphic imagingagent. This isotope has a single photon energy of 140 keV and aradioactive half-life of about 6 hours, and is readily available from a⁹⁹Mo-^(99m)Tc generator.

The metal radionuclides may be chelated by, e.g., linear, macrocyclic,terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also U.S. Pat. No.5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat.No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelators known inthe art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, TETA,and bisamino bisthiol (BAT) chelators (see also U.S. Pat. No.5,720,934). For example, N₄ chelators are described in U.S. Pat. Nos.6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487.Certain N₃S chelators are described in PCT/CA94/00395, PCT/CA94/00479,PCT/CA95/00249 and in U.S. Pat. Nos. 5,662,885; 5,976,495; and5,780,006. The chelator may also include derivatives of the chelatingligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains anN₃S, and N₂S₂ systems such as MAMA (monoamidemonoaminedithiols), DADS(N₂S diaminedithiols), CODADS and the like. These ligand systems and avariety of others are described in Liu and Edwards, Chem Rev, 1999, 99,2235-2268 and references therein.

The chelator may also include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. Nos. 5,183,653; 5,387,409; and 5,118,797, thedisclosures of which are incorporated by reference herein, in theirentirety.

Preferred metal chelators include those of FIGS. 8 from 8A to 8C (for¹¹¹In and lanthanides such as paramagnetic Gd³⁺ and radioactivelanthanides, such as, for example ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, and ¹⁶⁶Ho and thoseof FIGS. 9 from 9A to 9B (for radioactive ^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re).Particularly preferred metal chelators are those of formula 17-21 forGd³⁺ and radioactive lanthanides, and from 22 to 33 for radioactive^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re. These and other metal chelating groups aredescribed in U.S. Pat. Nos. 6,093,382 and 5,608,110; U.S. Pat. No.6,143,274; U.S. Pat. Nos. 5,627,286 and 6,093,382; U.S. Pat. Nos.5,662,885, 5,780,006, and 5,976,495 which are incorporated by referenceherein in their entirety. Additionally, the chelating formula 19 isdescribed in, for example, U.S. Pat. No. 6,143,274; the chelating groupof formula 31 and 32 are described in U.S. Pat. Nos. 5,627,286 and6,093,382; and the chelating group of formula 33 is described in, forexample, U.S. Pat. Nos. 5,662,885, 5,780,006, and 5,976,495.

In the above Formulae 17 and 18, R is alkyl, preferably methyl. In theabove Formulae 31 and 32 X is either CH₂ or O, Y is either C₁-C₁₀branched or unbranched alkyl; Y is aryl, aryloxy, arylamino,arylaminoacyl; Y is arylkyl—where the alkyl group or groups attached tothe aryl group are C₁-C₁₀ branched or unbranched alkyl groups, C₁-C₁₀branched or unbranched hydroxy or polyhydroxyalkyl groups orpolyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups, J is C(═O),OC(═O)—, SO₂, NC(═O)—, NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—,homopolyamides or heteropolyamines derived from synthetic or naturallyoccurring amino acids; all where n is 1-100. Other variants of thesestructures are described, for example, in U.S. Pat. No. 6,093,382. Thedisclosures of each of the foregoing patents, applications andreferences are incorporated by reference herein, in their entirety.

In a still further embodiment R₆ is an optionally labelled sugar moietyfor use, when labelled, in PET Imaging. In particular R₆ may represent asugar moiety labeled by halogenation with radionuclides, such as, ¹²⁴I,¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, and ⁷⁶Br wherein ¹⁸F is preferred.

Optical Imaging, Sonoluminescence or Photoacoustic Imaging

In one further preferred embodiment, R₆ represents a dye molecule; afluorescent molecule; a phosphoreent molecule; a molecule absorbing inthe UV spectrum; a quantum dot; or a molecule capable of absorption ofnear or far infrared radiations. Optical parameters to be detected inthe preparation of an image may include, e.g., transmitted radiation,absorption, fluorescent or phosphorescent emission, light reflection,changes in absorbance amplitude or maxima, and elastically scatteredradiation. For example, biological tissue is relatively translucent tolight in the near infrared (NIR) wavelength range of 650-1000 nm. NIRradiation can penetrate tissue up to several centimeters, permitting theuse of the diagnostic agents of the invention comprising a NIR moiety toimage target-containing tissue in vivo.

Near infrared dye may include, cyanine or indocyanine derivatives, suchas, for example, Cy5.5, IRDye800, indocyanine green (ICG), indocyaninegreen derivatives including the tetrasulfonic acid substitutedindocyanine green (TS-ICG), and combination thereof.

In another embodiment, the compounds of the invention may be conjugatedwith photolabels, such as optical dyes, including organic chromophoresor fluorophores, having extensively conjugated and hence delocalizedring systems and having absorption or emission maxima in the range of400-1500 nm. The compounds of the invention may alternatively bederivatized with a bioluminescent molecule. The preferred range ofabsorption maxima for photolabels is between 600 and 1000 nm to minimizeinterference with the signal from hemoglobin. Preferably,photoabsorption labels have large molar absorptivities, e.g. >105cm⁻¹M⁻¹, while fluorescent optical dyes will have high quantum yields.Examples of optical dyes include, but are not limited, to thosedescribed in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, andreferences cited therein. For example, the photolabels may be covalentlylinked directly to the targeting moieties of the invention, or they maybe linked thereto via a linking moiety, as described previously.

After injection of the optically-labeled diagnostic derivative accordingto the invention, the patient is scanned with one or more light sources(e.g., a laser) in the wavelength range appropriate for the photolabelemployed in the agent. The light used may be monochromatic orpolychromatic and continuous or pulsed. Transmitted, scattered, orreflected light is detected via a photodetector tuned to one or multiplewavelengths to determine the location of target-containing tissue (e.g.angiogenic tissue) in the subject. Changes in the optical parameter maybe monitored over time to detect accumulation of the optically-labeledreagent at the target site. Standard image processing and detectingdevices may be used in conjunction with the optical imaging reagents ofthe present invention.

The optical imaging agents described above may also be used foracousto-optical or sonoluminescent imaging performed withoptically-labeled imaging agents (see, f.i., U.S. Pat. No. 5,171,298, WO98/57666, and references therein). In acousto-optical imaging,ultrasound radiation is applied to the subject and affects the opticalparameters of the transmitted, emitted, or reflected light. Insonoluminescent imaging, the applied ultrasound actually generates thelight detected. Suitable imaging methods using such techniques aredescribed in WO 98/57666, which is hereby incorporated by reference inits entirety.

Special Purpose Molecules

In a still further embodiment R₆ is a special purpose molecule. Asspecial purpose molecule are defined some molecules that may or may nothave a direct role in the interaction with a detection system, but havea functional attribute that facilitates the use of the peptidicconstructs in some way. Such molecules, when appended to the peptidicconstruct, for example, may enable the incorporation of the peptidicconstruct into larger arrays of molecules (such as supramolecularconstructs) which can conversely be detected by equipment or apparatusemployed for detection of signals, whether by presence or absence of thesignal, a change in signal intensity, reflection of a signal or aderivative signal during or after a period of irradiation by theequipment or apparatus.

Thus, in one embodiment, R₆ is a phospholipid or lipid moiety consentingto the cyclic npeptides of the invention linked thereto to beincorporated, iiupon agitation (e.g., shaking, stirring, etc.) intoliposomes, or even micellar systems, vesicles or microspheres suitablyincluding, for example, metal entities and particularly, paramagneticmetal ions, or an echogenic gas, thus providing a macromolecularcompound for use in MRI or ultrasound imaging including an high numberof the said targeting moiety on its surface. Together with thepeptidomimetic compounds of the invention comprising a lipid moietythese macromolecular system may further include surfactants,sphingolipids, oligolipids, phospholipids, proteins, polypeptides,carbohydrates, and synthetic or natural polymeric materials. See, e.g.,WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18497, WO 98/18496, and WO98/18501 incorporated herein by reference in their entirety.

A “lipid” as used herein, is a synthetic or naturally-occurringamphipatic compound which comprises a hydrophilic component and ahydrophobic component. Lipid includes, for example, fatty acids, neutralfats, phosphatides, glycolipids, aliphatic alcohols and waxes, terpenesand steroids.

Examples of classes of suitable lipids constituting the R₆ moiety of theinvention include: phosphatidylcholines, such asdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipalmitoyl-phosphatidylcholine and diasteroylphosphatidylcholine;phosphatidyl-ethanolamines, such as dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine andN-succinil-dioleoylphosphatidyl-ethanolamine; phosphatidylserine;dipalmitoylphosphatidylserine; phosphatidylglycerols; sphingolipids;glycolipids such as ganglioside GMvil; glucoiips; suiphatides;phosphatidic acid, such as dipalmitoyl phosphatidic acid (“DPPA”);palmitic fatty acids; stearic fatty acids; arachidonic fatty acids;lauric fatty acids; myristic fatty acids; lauroleic fatty acids;physeteric fatty acids; myristoleic fatty acids; palmitoleic fattyacids; petroselinic fatty acids; oleic fatty acids; isolauric fattyacids; isomyristic fatty acids; isostearic fatty acids; cholesterol andcholesterol derivatives, such as cholesterol hemisuccinate, cholesterolsulphate, and cholesteryl-(4-trimethylammonio)-butanoate;polyoxyethylene fatty acids asters, polyoxyethylene fatty acidsalcohols; polyoxyethylene fatty acids alcohol ethers; polyoxyethylatedsorbitan fatty acid esters, glycerol polyethylene glycol oxy-stearate;glycerol polyethylene glycol ricinoleate; ethoxylated soybean sterols;ethoxylated castor oil; polyoxyethylene polyoxypropylene fatty acidpolymers; polyoxyethylene fatty acid stearates;1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoyl-glycerophosphoethanolamina;N-Succinyl-dioctadecylamine; palmitoylhomocysteine;lauryltrimethylammonium bromide; cetyltrimethyl-ammonium bromide;myristyltrimethylammonium bromide; alkyldimethylbenzylammonium chloride;wherein alkyl is a C₁₂, C₁₄, C₁₆ alkyl; benzyldimethyldodecylammoniumbromide; benzyldimethyldodecyl ammonium chloride;benzyldimethylhexadecylammonium bromide; benzyldimethylhexadecylammoniumchloride; benzyldimethyltetradecyl ammonium bromide;benzyldimethyltetradecyl ammonium chloride; cetyldimethylethylammoniumchloride; cetylpyridinium bromide; cetylpyridinium chloride;N-[1,2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA);1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); and1,2-dioleoyl-c-(4′-trimethylammonium)-butanoyl-sn-glycerol (DOTB).

With the term “vesicle”, as used herein, we refer to a spherical entitywhich is characterized by the presence of an internal void. Preferredvesicles are formulated from lipids, including the various lipidsdescribed herein. In any given vesicle, the lipids may be in the form ofmonolayer or bilayer, and the mono-or bilayer lipids may be used to formone or more of mono-or bilayers. The lipid vesicles described hereininclude such entities commonly referred to as liposomes, micelles,bubbles, microbubbles, microspheres and the like. The internal void ofthe vesicles may be filled with a liquid, including, for example, anaqueous liquid, a gas, a gaseous precursor, and/or a solid or solutematerial, including for example, a bioactive agent.

Liposomes, as used herein, refers to a generally spherical cluster oraggregate of amphipatic compounds, including lipid compounds, typicallyin the form of one or more concentric layers, for example, bilayers.They may also be referred to herein as lipid vesicles.

The term bubbles as used herein refers to a vesicles which are generallycharacterized by the presence of one or more membranes o wallssurrounding an internal void that is filled with a gas or a precursorthereto. Exemplary bubbles includes, for example, micelles, liposomes,and the like.

Microspheres as used herein, is preferably a sphere of less than orequal to 10 microns.

Thus, in one embodiment the invention relates to a macromolecular systemin the form of liposomes, micelles, microemulsions, bubbles,microbubbles, microballons or microspheres comprising a compound of theinvention wherein R₆ is a phospholipid or a lipid moiety.

In one preferred embodiment, the said macromolecular system is aparamagnetic liposome, i.e. a liposome containing in its cavity Gd³⁺ions, for use in MRI of integrin α_(v)β₃ expression.

In a further preferred embodiment the said macromolecular system is aLIPOCEST.

In another preferred embodiment, the said macromolecular system is a gascontaining vesicle for use in ultrasound imaging.

In an additional embodiment the said gas filled vesicles aregas-containing microparticle.

Ultrasound Contrast Agents,

in a further embodiment R₆ represents any material includingsurfactants, sphingolipids, oligolipids, phospholipids, proteins,polypeptides, carbohydrates, and synthetic or natural polymericmaterials coupled to a peptidic derivative incorporated into a gasfilled vesicle, i.e. a microbubbles, microballoons, microspheres oremulsions containing a liquid or gas moiety which functions as thedetectable label (e.g., an echogenic gas or material capable ofgenerating an echogenic gas).

Preferably, R₆ represents, a phospholipid or lipid moiety according tothe above definition coupled to a peptidic derivative incorporated intoa gas filled vesicle.

In one embodiment the said gas filled vesicles are bubbles ormicrobaloons.

The preferred microballoons have an envelope including a biodegradablephysiologically compatible polymer or a biodegradable solid lipid. Thepolymers useful for the preparation of the microballoons of the presentinvention can be selected from the biodegradable physiologicallycompatible polymers, such as any of those described in any of thefollowing patents: EP 458745, U.S. Pat. No. 5,711,933, U.S. Pat. No.5,840,275, EP 554213, U.S. Pat. No. 5,413,774 and U.S. Pat. No.5,578,292, the entire contents of which are incorporated herein byreference. In particular, the polymer can be selected from biodegradablephysiologically compatible polymers, such as polysaccharides of lowwater solubility, polylactides and polyglycolides and their copolymers,copolymers of lactides and lactones such as ε-caprolactone,γ-valerolactone and polypeptides. Other suitable polymers includepoly(ortho)esters (see, e.g., U.S. Pat. No. 4,093,709; U.S. Pat. No.4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646); polylacticand polyglycolic acid and their copolymers, for instance DEXON (see J.Heller, Biomaterials 1 (1980), 51; poly(DL-lactide-co-ε-caprolactone),poly(DL-lactide-co-γ-valerolactone),poly(DL-lactide-co-γ-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; poly-dioxanone; poly-β-aminoketones (A.S. Angeloni, P. Ferruti, M. Tramontini and M. Casolaro, The Mannichbases in polymer synthesis: 3. Reduction of poly(beta-aminoketone)s topoly(gamma-aminoalcohol)s and their N-alkylation topoly(gamma-hydroxyquaternary ammonium salt)s, Polymer 23 pp 1693-1697(1982)); polyphosphazenes (Aiicock, Harry R., Polyphosphazenes: newpolymers with inorganic backbone atoms (Science 193(4259), 1214-19(1976)) and polyanhydrides. The microballoons of the present inventioncan also be prepared according to the methods of WO-A-96/15815,incorporated herein by reference, where the microballoons are made froma biodegradable membrane comprising biodegradable lipids, preferablyselected from mono- di-, tri-glycerides, fatty acids, sterols, waxes andmixtures thereof. Preferred lipids are di- or tri-glycerides, e.g. di-or tri-myristin -palmityn or -stearin in particular tripalmitin ortristearin.

The microballoons may employ any of the gases disclosed herein or knownto the skilled artisan.

Any biocompatible gas may be used in the vesicular contrast agents ofthe invention.

The term “gas” as used herein includes any substances (includingmixtures) substantially in gaseous form at the normal human bodytemperature.

The said gas may thus include, for example, air; nitrogen; oxygen; CO₂;argon; xenon or krypton, fluorinated gases (including for example,perfluorocarbons, SF₆, SeF₆) a low molecular weight hydrocarbon (e.g.containing from 1 to 7 carbon atoms), for example, an alkane such asmethane, ethane, propane, butane or pentane, a cycloalkane such ascyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene,propene, propadiene or butene, or an alkyne such as acetylene or propyneand/or mixtures thereof. However, fluorinated gases are preferred.Fluorinated gases include materials which contain at least one fluorineatom. Examples include, but are not limited to compounds such as SF6,freons (organic compounds containing one or more carbon atoms andfluorine, i.e. CF₄, C₂F₆, C₃Fs, C₄F₈, C₄F₁₀, CBrF₃, CCI₂F₂, C₂CIF₅ andCBrClF₂) and perfluorocarbons. The term perfluorocarbon refers tocompounds containing only carbon and fluorine atoms. Such compoundsinclude saturated, unsaturated, and cyclic perfluorocarbons. Thesaturated perfluorocarbons, which are preferred, have the formulaC_(n)F_(n+2), where n is from 1 to 12, preferably from 2 to 10, morepreferably from 3 to 8 and most preferably from 3 to 6. Suitableperfluorocarbons include, but are not limited to, CF₄, C₂F₆, C₃F₈, C₄F₈,C₄F₁₀, C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈ and C₉F₂₀. Most preferably, the gas orgas mixture comprises SF₆ or a perfluorocarbon selected from the groupconsisting of C₃F₈, C₄F₈, C₄F₁₀, CF₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, with C₄F₁₀being particularly preferred.

In certain circumstances it may be desirable to include a precursor to agaseous substance (e.g., a material that is capable of being convertedto a gas in vivo, often referred to as a “gas precursor”). Preferably,the gas precursor and the gas it produces are physiologicallyacceptable. The gas precursor may be pH-activated, photo-activated,temperature-activated, etc. For example, certain perfluorocarbons may beused as temperature-activated gas precursors. These perfluorocarbons,such as, e.g., perfluoropentane, have a liquid/gas phase transitiontemperature above room temperature (or the temperature at which theagents are produced and/or stored) but below body temperature; thus,they undergo a phase shift and are converted to a gas within the humanbody.

In one further embodiment, R₆ is a biotin moiety or a biotinylatedmoiety consenting to a cyclic peptides of the invention linked theretoto be appended to a suitably functionalized macromolecular aggregatethat comprise, in its internal void, for example, metal entities or anechogenic gas, thus providing a detectable multifunctionalizedmacromolecular aggregate.

The multifunctionalized compounds obtained exploiting the said specialpurpose R₆ moieties comprise an high number of targeting units of theinvention on thereof surface. Accordingly, they may optimally bind acell surface or an intravascular surface expressing the targetedreceptor and, when successfully bound to the angiogenic targetselectively addressed by the peptidic moieties they comprise, then thesaid derivative may be detected by use of a suitable imaging techniquedepending on what the vesicle cavity has been filled, thereby allowingdiagnosis or prognosis of a disease condition.

Liposomes, for example, may also be employed to facilitate delivery ofencapsulated solutions of chemotherapeutic agents. In this case, thephospholipid-peptide conjugates of the invention can be incorporatedinto liposomes, thereby enabling the liposome to target a specificintegrin receptors and release the therapeutic in higher concentrationand proximity to the targeted region.

Suitable examples or the this multifunctionalized system are preparedaccording to known techniques (see, e.g., Sipkins, D. A. et al., 1998,Nat. Med. 4:623-626 and cited references). Best disclosure of thesecompounds and thereof preparations are comprised in the experimentalsection below.

Divalent Linking Moiety

With the term “divalent linking moiety”, when referring to (L) informula (III), we intend a bifunctional moiety including at least twobinding groups for the attachment with the remaining portions of themolecule, for instance through cross-linking or coupling reactions.

Typically, any linking moiety (L) may be represented through thedifunctional groups from (i) to (v) or it can be represented by adivalent linking moiety connecting R₆ to the —(CH₂)p-bicyclo derivativein formula (III), comprising two of the above (i) to (v) groups, asending groups.

As a non limiting example, within a compound of formula (III) beingprepared as schematically indicated below:

R₆—COOH+H₂N-(chain)-COOH+H₂N—(CH₂)p-bicyclo-------->------->R₆—CONH-(chain)-CONH—(CH₂)p-bicyclo

the linking moiety (L) is just the divalent moiety in bold, connectingR₆ to the —(CH₂)p-bicyclo residue, this latter herewith shortly referredto as the targeting moiety or unit.

Besides acting as a branching system between R₆ and the targeting unit,the linking moiety L may provide for a proper distance or “space”between the biologically active moiety R₆ and the targeting unit of thecompound of the invention. In fact, an optimal distance between thesetwo units may represent an important factor so as to get and maintainthe targeting capability of the cyclic peptide of the invention. It iswell known, in fact, that any improper or anyway sub-optimalderivatization of a peptidic-based targeting moiety may often result ina significant loss of the peptide affinity for the targeted objective.

Moreover, in a further and equally relevant view of the invention, thelinkers (L) may significantly contribute to improve the hydrophilicityof the diagnostic or therapeutic agent, thus providing the desiredpharmacokinetic or pharmacodynamic profile of the compound of formula(III).

Accordingly, L may include, without limitations: substituted orunsubstituted, either saturated or unsaturated, straight or branchedalkyl chains; pepntidP frnm s-raight, branched or cyclic amino acidchains composed from a single amino acid or from different amino acids(e.g., extensions of the N- or C-terminus of the binding moieties);derivatized or underivatized polyethylene glycol, polyoxyethylene, orpolyvinylpyridine chains; substituted or unsubstituted polyamide chains;derivatized or underivatized polyamine, polyester, polyethylenimine,polyacrylate, poly(vinyl alcohol), polyglycerol, or oligosaccharide(e.g., dextran) chains; glycosylated amino acid residues, alternatingblock copolymers; malonic, succinic, glutaric, adipic and pimelic acids;caproic acid; simple diamines and dialcohols; any of the other linkersdisclosed herein; or any other simple polymeric linker known in the art(see, e.g., WO 98/18497, WO 98/18496).

From the above definitions, it is clear to the skilled person that incase L includes an alkyl chain, for instance a propylene chain —(CH₂)₃—,the divalent linking moiety L in formula (III) could be represented,just as an example, as

—CONH—(CH₂)₃—NHCONH—

said group bridging the bioactive moiety R₆ through the carboxamidogroup, at one side, and the targeting unit —(CH₂)p-bicyclo through theureido group, at the other side.

Likewise, for instance in the case of L including an amino acid, forexample Ala, the divalent linking group L in formula (III) could berepresented, as an example, as

—CONH—CH(CH₃)CONH—

wherein R₆ may be connected, via carboxamido, to the amino group of Ala,whilst the targeting unit may be connected, in the present case throughcarboxamido, to the carboxy group of Ala.

The molecular weight (MW) of the linking moieties L of the invention maybe selected, for instance up to about 1000 dalton, preferably up toabout 500 and even more preferably up to about 300 dalton.

In addition, it may be desirable to utilize a linking moieties that isbiodegradable in vivo so as to provide efficient routes of excretionupon administration of the compounds of the invention, for instance of adiagnostic agent.

Depending upon their location within the linking moieties itself,suitable biodegradable groups can be thus present. Typically, abiodegradable functionality may include an ester, double ester, amide,phosphoester, ether, acetal and ketal functionality.

As an additional example, the linking moiety of the invention may alsoinclude homobifunctional or heterobifunctional moieties or suitablecombinations thereof.

With the term homobifunctional molecule or moiety, as used herein, weintend a molecule or moiety having at least two reactive functionalgroups which are the same. With the term heterobifunctional molecule ormoiety, as used herein, we intend a molecule or moiety having at leasttwo different reactive groups.

Suitable examples of homobifunctional molecule include, for example,dicarboxylic compounds and suitable derivatives thereof wherein thecarboxylic group(s) are in a suitably activated or protected form; adiamine and suitable derivatives thereof wherein the amino group(s) arein a suitably activated o protected form.

Examples of dicarboxylic molecule include the following compounds:

HOOC—Z—COOH where Z can be in the form (CH₂)_(n) to give compounds suchas, e.g, HOOC—(CH₂)_(n)—CO₂H where n=0-10. The group Z can also be analkyl chain which is mono or polysubstituted by one or more of thefollowing groups: —NH—, —O—, —CO—, —NH(CO)—, —(CO)NH—, —O(CO)—, or—(OC)O— thus resulting in structures like, for instance:

-   -   HOOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—COOH    -   HOOC—CH₂—NH—CH₂—COOH    -   HOOC—(CH₂)₂—CO₂—(CH₂)₂—OCO—(CH₂)₂—COOH    -   HOOC—CH(OH)—CH(OH)—COOH        In a further embodiment, the said dicarboxylic molecules can        also comprise a substituted aromatic or heterocyclic acid of the        form HOOC—Z—COOH, where Z represents the aromatic or        heterocyclic nucleus which is the scaffold bearing the two        carboxylic groups. Examples of such molecules include, without        limitation, benzene-1,4-dicarboxylic acid,        hiphenyl-1,4′-dicarboxylic acid,        (4-carboxymethoxy-phenoxy)-acetic acid,        (4′-carboxymethoxy-biphenyl-4-yloxy)-acetic acid,        N-methylpyrrole-2,5-dicarboxylic acid, pyridine-2,6-dicarboxylic        acid, N-methylpyrrole-2,3-dicarboxylic acid,        N-methylpyrrole-2,4-dicarboxylic acid, pyridine 2,3-dicarboxylic        acid, pyridine-3,5-dicarboxylic acid, pyridine 3,4-dicarboxylic        acid or piperidine 3,5-dicarboxylic acid. The saturated        derivatives of the aromatic compounds described above, where Z        is now a saturated derivative of the aromatic or heterocyclic Z,        can also be employed to provide suitable linkers L according to        the present invention.

Examples of homobifunctional diamine molecules include the following:

-   -   NH₂—(CH₂)_(n)—NH₂, where n=0-20; or

NH₂—CH₂(CH₂)_(j)O—(CH₂(CH₂)_(m)O)_(n)—CH₂—(CH₂)_(p)—NH₂, where j=1-20,m=1-2, n=1-100 and p=1-20.

According to an additional embodiment, the linking moieties L may bederived from a substituted aromatic or heterocyclic diamine of the formNH₂-Q-Z-Q′—NH₂, where Z represents the aromatic or heterocyclic nucleuswhich is the scaffold bearing the two amino groups; and Q and Q′ are:

-   -   —(CH₂)_(n) where n=2-10; or    -   —CH₂(CH₂)_(f), where f=1-9; or    -   —(CH₂CH₂O)_(q)(CH₂)_(r), where q=1-10 and r=2-10; or    -   —(CH₂)_(n)NH—C(═O)— where n=2-10; or    -   —(CH₂CH₂O)_(q)(CH₂)_(r)—NH—C(═O)— where q=1-10 and r=2-10;        and where Q and Q′ can be the same or different members of the        set of moieties described herein.

Clearly, when referring to dicarboxylic or diamine molecules ormoieties, previous considerations to the linking group (L) in formula(III) apply as well. Therefore, when referring to a dicarboxylic acidlike

HOOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—COOH

a divalent linking moiety L could be represented in formula (III), forinstance as R₆—NHCO—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—CONH—(CH₂)p-bicyclo or,even R₆—CONHCO—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—CONH—(CH₂)p-bicyclo.

Analogously, any diamine like

NH₂—(CH₂)_(n)—NH₂

could give rise, in formula (III), to a divalent linking moiety forinstance as:R₆—CONH—(CH₂)_(n)—NHCONH—(CH₂)p-bicyclo orR₆—NHCONH—(CH₂)_(n)—NHCONH—(CH₂)p-bicyclo orR₆—NHCONH—(CH₂)_(n)—NHCSNH—(CH₂)p-bicyclo, and the like.

In a further embodiment, the linking moiety of the invention maycomprise a suitable mono or bis-imide derivative of the aforementionedbis-amines, wherein maleimido derivatives are particularly preferred.

Examples of heterobifunctional linking moieties may include compounds atleast bearing an amino and a carboxylic function as reactive groups.

In a preferred embodiment, the said amino acid molecules are derivedfrom a D or L amino acid, including, as a non limiting example, glycine,lysine, serine, ornithine, 2,3-diaminopropionic acid, or a suitablecombination thereof.

In another embodiment, the said amino acids may be suitably glycosylatedthus enhancing solubility and binding capability with integrin viaaspecific or non-specific interactions involving the glycosydic moiety.

Accordingly, L may optionally include one or more sugar moiety suitablyselected from the following: N-acetylgalactoseamine, D-(+)-allose,D-(+)-altrose, D-(+)-glucose, D-(+)-mannose, D-(−)-gulose, D-(−)-idose,D-(+)-galactose, D-(−)-talose, D-(−)-ribose, D-(−)-arabinose,D-(+)-xylose or D-(−)-lyxose for example, linked to a serine, threonineor asparagines residue, and, preferably, serine, through thereofside-chain oxygen via the anomeric carbon.

For additional definitions of possible linking moieties according to theinvention see, as an example, the following sections related to themanufacturing process.

The term “pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds of the invention wherein the parentcompound is modified by making the acid or basic groups not yetinternally neutralized in the form of non-toxic, stable salts which doesnot destroy the pharmacological activity of the parent compound.Suitable example of the said salts include: mineral or organic acidsalts, of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like.

Preferred cations of inorganic bases which can be suitably used tosalify the compounds of the invention comprise ions of alkali oralkaline-earth metals such as potassium, sodium, calcium or magnesium.

Preferred cations of organic bases comprise, inter alia, those ofprimary, secondary and tertiary amines such as ethanolamine,diethanolamine, morpholine, glucamine, N-methylglucamine,N,N-dimethylglucamine.

Preferred anions of inorganic acids which can be suitably used to salifythe complexes of the invention comprise the ions of halo acids such aschlorides, bromides, iodides or other ions such as sulfate.

Preferred anions of organic acids comprise those of the acids routinelyused in pharmaceutical techniques for the salification of basicsubstances such as, for instance, acetate, succinate, citrate, fumarate,maleate or oxalate.

Preferred cations and anions of amino acids comprise, for example, thoseof taurine, glycine, lysine, arginine, ornithine or of aspartic andglutamic acids.

The pharmaceutically acceptable salts of the invention may be preparedfrom the parent compound which contain a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting the free base or acid forms of these compounds with astoichiometric amount of the appropriate base or acid in an organicsolvent or in a mixture of the two.

Multimeric Constructs

In a further embodiment, the present invention relates to novelmultimeric constructs including two or more active moieties linked to ananchoring system. Suitable example of the said active moieties include:a targeting moiety, a diagnostically active moiety, a therapeutic agent,a drug, a sugar, a lipid, a biotin residue.

In particular, in a further aspect, the invention relates to novelmultimeric targeting constructs that can exploit the avidity andspecificity of multivalent interactions with targeted αvβ3 integrins byincluding at least two cyclic peptides of the invention, an anchoringsystem and optional linkers and to novel diagnostic or therapeuticcompounds including them.

With the term “anchoring system”, as used herein, we intend apolifunctional compound containing at least three, optionally protected,equal or different binding sites or functional groups deriving from anypolyvalent organic residue which can be aliphatic with open chain,optionally branched, or alicyclic, or heterocyclic containing N,O,and/or S or aromatic or heteroaromatic, or it is a streptavidin oravidin moiety.

The terms “functionality” or “functional group”, are used hereininterchangeably to refer to specific groups of atoms within molecules ormoieties that are responsible for the characteristic chemical reactionsof those molecules or moieties. In the context of the present invention,the functional group is the specific active part of a binding groupallowing cross-linking or coupling reactions.

With the terms “binding group” or “binding unit” or “branching group” asinterchangeably used herein we intend a group or unit able to chemicallyreact with a second, suitable, group thus resulting in the chemicalconjugation or “binding” thereof.

With the term “protecting group”, as used herein, we designates aprotective group adapted to preserving the function to which it isbound. Specifically, protective groups are used to preserve aminofunction or carboxyl function. Appropriate protective groups include forexample benzyl, benzyloxycarbonyl, alkyl or benzyl esters, or othersubstituents commonly used for the protection of such functions, whichare well known to those skilled in the art, for example those describedin conventional manuals such as T. W. Green, Protective Groups inOrganic Synthesis (Wiley, N.Y. 1981).

A “multimeric targeting construct”, as used herein, is a multimericconstruct comprising two or more cyclic peptide of the invention thatthus can exploit the avidity and specificity of multivalent interactionswith targeted αvβ3 integrins.

With the term “construct” or “multimeric construct” as used hereininterchangeably, we intend a multimeric or oligomeric derivative of anactive moiety that includes at least two of the said moieties suitablylinked to an anchoring system.

It is therefore an additional object of the invention a compound offormula (IV)

wherein

R₄, R₅, n, p, and L, equal or different among them, have the meaningsabove reported for the compounds of formula (III),

T is an anchoring system, and

r is an integer from 2 to 10,

their salts, racemic mixtures, individual enantiomers, individualdiastereoisomers and mixtures thereof in whatever proportion.

Preferably, within the compounds of formula (IV), r is an integer from 2to 5.

Suitable examples of anchoring (T) systems according to the inventioninclude:

-   (a) N-branched lysine systems (see, f. i., Veprek, P et al., J.    Pept. Sci. 5, 5 (1999); 5, 203 (1999),-   (b) polycarboxylic derivatives,-   (c) polyaminated derivative,-   (d) amino acids,

Non limiting examples of preferred anchoring systems according to theinvention are included in FIGS. 7A, 7B and 7C. All of these compoundsare well known in the art and most of them are already marketed. Thenon-marketed compounds may be easily prepared according to knownmethods, for example as per the accompanying bibliographic references.The compounds from 20 to 26 are prepared according references cited intoEP 1259532.

It is understood to those skilled in the art that lysine derivatives,ornithine, or 2,3-diamino propionic acid may be serially employed toelongate and/or to suitably increase the multiplicity of the saidmultimers.

An alternative embodiment is represented by the above multimericconstructs wherein the anchoring system (T) is a streptavidin or anavidin/biotin system.

In this respect, it is well known to those skilled in the art, that eachavidin molecule or moiety binds, selectively, four biotin molecules ormoieties. Thus, for example, an avidin molecule may act as an anchoringsystem able to bind three units of a biotinylated targeting peptide ofthe invention, i.e., for example, a compound of formula (III) in whichR₆ is, or includes, a biotin moiety.

It is therefore an additional object of the invention a compound offormula (V)

wherein

R₄, R₅, n, p and L, equal or different among them, have the meaningsformerly reported for the compounds of formula (III),

R₆ is a biotin moiety,

T is a streptavidin or an avidin moiety,

r is 2 or 3,

their salts, racemic mixtures, individual enantiomers, individualdiastereoisomers and mixtures thereof in whatever proportion.

Preferably, within the compounds of formula (V), r is 3.

The compounds of formulae (IV) and (V) possess interestingpharmacological properties, particularly an antagonistic effect towardsthe αvβ3 and αvβ5 integrins, and display interesting antiangiogenicactivities.

A further object of the present invention is hence the use of thesecompounds for the preparation of drugs, particularly useful for theirantagonistic action towards the αvβ3 and αvβ5 integrins.

More particularly, the invention concerns the use of compounds ofgeneral formulae (IV) and (V) for the preparation of drugs useful forthe treatment of both altered angiogenic phenomena, and for those thatmay be encountered in metastasising tumour processes, retinopathies,acute renal damage and osteoporosis.

In one preferred embodiment of the invention, the anchoring system (T)binds with at least two targeting units of the invention and with atlest one biologically active molecule or moiety R₆, optionally throughdivalent linking moieties.

Therefore, it is a further object of the invention a compound of formula(VI)

wherein

R₄, R₅, n, p, T and L, equal or different among them, have the meaningsformerly reported for the compound of formula (IV);

r is an integer from 2 and 10, and

R₆ is a biologically active moiety,

their salts, racemic mixtures, individual enantiomers, individualdiastereoisomers and mixtures thereof in whatever proportion.

In a different aspect, the anchoring system (T) of the invention maybind, optionally through linking moieties, to two or more biologicallyactive molecules according to the invention thus providing a multimericconstruct showing improved biological activity.

Thus, in a still further embodiment the invention relates to novelcompounds of formula (VII)

in which:

R₄, R₅, n, p, T and L, equal or different among them, have the meaningsformerly defined for compounds of (IV),

b is an integer from 2 to 5, and

R₆ is a biologically active moiety,

their salts, racemic mixtures, individual enantiomers, individualdiastereoisomers and mixtures thereof in whatever proportion.

Moreover, the multimeric targeting derivatives of formula (V) still haveat least one free, optionally protected, binding site able to furtherbind, a biotin residue or a suitably biotinylated biologically activemolecule.

It is therefore a further object of the invention a macromolecularaggregate such as, for example, a vescicle, a microsphere, a micelle ora liposome moiety, each of which comprising a high number of biotinresidues on their surface, the said biotin residues being coupled, oranyway connected, with the above multimeric targeting constructs offormula (V).

Alternatively, these macromolecular aggregate may be obtained bycoupling the said biotinylated vescicles, microspheres, micelles or aliposomes with a compound of formula (III) wherein R₆ is biotin moietysuitably coupled to an avidin moiety.

These aggregates may be prepared according to conventional methods knownin the art, for instance as per the experimental section.

As an example, the said macromolecular aggregates can be represented byformula (VIII)

wherein:

R₄, R₅, n, p and L have the meanings above reported,

R₆ is a biotin moiety,

T is a streptavidin or an avidin moiety,

r is 2 or 3,

A is a vesicle, a microsphere, a micelle or a liposome moiety comprisinga number of biotin units on their surface,

s represents the number of the compounds of formula (V) permacromolecular aggregate.

Preferably the said s value is expressed as percent the saidbiotinylated peptidic derivative represents, being 100 the globalcomponents amount, including, surfactants, sphingolipids, oligolipids,phospholipids, proteins, polypeptides, carbohydrates, and synthetic ornatural polymeric materials amounts.

Preferably, within the compounds of formula (VIII), r is 3.

Preferably, within the compounds of formula (VIII), s is up to 10%, morepreferably up to 3 and most preferably s is 1%.

Suitable examples of compounds of formula (VIII) comprise macromolecularaggregates wherein A is represented by a paramagnetic liposome, aLIPOCEST or an echogenic gas filled vesicular compound.

In one embodiment, A in formula (VIII) comprises a biotinylatedvesicular compound filled with an echogenic gas.

In another embodiment A in formula (VIII) is a biotinylated paramagneticor superparamagnetic particle.

In a particularly preferred embodiment, A in formula (VIII) comprises abiotinylated LIPOCEST.

With LIPOCEST, as used herein, we intend a paramagnetic liposomes thatact as CEST agents (LIPOCEST agents) for use in CEST imaging protocols.

With CEST imaging as used herein, we relates to the generation ofcontrast in an MRI imaging through irradiation of mobile protons in aCEST contrast agent containing at least one mobile proton in exchangewith water or in a suitable CEST imaging system. In the presentinvention the CEST imaging system is represented by a liposomal system.In this case the chemical shift of the intraliposomal water protonswhich must be irradiated to observe saturation transfer has beensuitably “shifted” as a result of their interaction with a paramagneticchelate containing a metal selected from iron (II) (high-spinconfiguration), iron (III), cobalt (II), rhodium (II), copper (II),nickel (II), cerium (III), praseodymium (III), neodymium (III),gadolinium(III), dysprosium (III), erbium (III), terbium (III), holmium(III), thulium (III), ytterbium (III) and europium (III).

The paramagnetic complex can be encapsulated in the aqueous cavity ofthe liposome (if hydrophilic), and/or incorporated in the lipidicbilayer of the membrane (if amphiphilic).

The chemical shift difference between the resonances of intraliposomaland bulk water protons (Δ^(LIPO)) is dependent on the formulation andpreparation of the liposomes as well as the physico-chemical propertiesof the paramagnetic complex. In particular, the chemical shift of thewater proton is affected by: i) the concentration of the hydrophilicparamagnetic complex in the aqueous cavity (if encapsulated) and/or theconcentration of the paramagnetic complex incorporated in the membraneand facing the aqueous inner cavity of the liposome, and ii) theliposome shape. Macromolecular aggregates comprising a biotinylatedLIPOCEST, their preparation and characterization are best detailed inthe experimental section below.

Those skilled in the art may understand that macromolecular aggregatesaccording to the invention comprising a number of peptidomimeticmoieties on their surface may equally be obtained by connecting amacromolecular compound such as, for example, a vesicle, a microsphere,a micelle or a liposome including an high number of streptavidin oravidin moieties on their surface with a suitable number of biotinylatedtargeting derivatives of the invention, i.e., for example, compounds offormula (III) wherein R₆ is or comprises a biotin residue.

All the compounds of each of the formulae from (III) to (VIII) moreoverpossess interesting pharmacological properties, particullarly anantagonistic effect towards the αvβ33 and αvβ5 integrins, and displayinteresting antiangiogenic activities.

Thus, according to another aspect thereof, the invention concernspharmaceutical compositions containing, as active ingredient, at leastone compound of each of the formulae from (III) to (VIII), thepharmaceutically acceptable salts, racemic mixtures, individualenantiomers, individual diastereoisomers and mixtures thereof inwhatever proportion, in combination with one or more possiblepharmaceutically acceptable carriers or excipients.

In a further aspect, the invention concerns a contrast agent accordingto anyone of formulae (III), (V), or (VIII) wherein R₆ is an imagingdetectable moiety or in the form of imaging detectable macromolecularaggregate comprising on their surface a number of targeting moietiesaccording to the invention.

In one embodiment the said contrast agent is an MRI contrast agent, and,preferably, is a paramagnetic liposomes that act as CEST agents.

Preparations

The novel compounds of the invention may be prepared by starting, atfirst, from the compounds of formula (I) below

where:

n is 1 or 2,

p is an integer between 1 and 5,

R₁ is H, (C₁-C₄) alkyl or a protective group,

R₂ is H or a protective group,

X is —N₃, —NHR₃, —SR₃, —N═C═O, or —N═C═S,

wherein R₃═H or a protective group;

their salts, racemic mixtures, individual enantiomers, individualdiasteroisomers and mixtures thereof in any portion.

The compounds of formula (I) may exist in different configurations

where n, R₁, R₂ and X are as defined above and the wedge-shaped anddashed bonds indicate that the substituents are positioned above andbelow the plane respectively. Unless otherwise provided, the term“(C₁-C₄) alkyl” designates a linear or branched, saturated orunsaturated alkyl substituent comprising from 1 to 4 carbon atoms suchas for example methyl, ethyl, propyl, isopropyl, butyl, tert-butyl.However, it is possible to use alkyl substituents containing a highernumber of carbon atoms providing they are compatible with the reactionconditions of the present invention. According to the present invention,the expression “protective group” designates a protective group adaptedto preserving the function to which it is bound, specifically the aminofunction or carboxyl function. Appropriate protective groups include forexample benzyl, benzyloxycarbonyl, alkyl or benzyl esters, or othersubstituents commonly used for the protection of such functions, whichare well known to those skilled in the art, for example those describedin conventional manuals such as T. W. Green, Protective Groups inOrganic Synthesis (Wiley, NY 1981).

The salts of the compounds of formulae (I), (Ia) and (Ib), according tothe present invention, comprise both those with mineral or organic acidsand those forming physiologically and pharmaceutically acceptable salts,such as for example hydrochloride, hydrobromide, sulphate, hydrogensulphate, dihydrogen sulphate, maleate, fumarate,2-naphthalenesulphonate, par_(a)-toluenesulphonate, oxalate etc.

Salts of the compounds of formulae (I), (Ia) and (Ib) according to thepresent invention also further include physiologically andpharmaceutically acceptable quaternary ammonium salts.

Said salts are prepared according to the well known techniques for theperson skilled in the art.

When there is a free carboxyl group (R₂═H) present, the salts of thecompounds of formula (I) also comprise salts with organic or mineralbases, such as for example alkaline metal or alkaline earth metal salts,such as sodium salts, potassium or calcium salts, or with an amine suchas trometamol (tromethamine), or salts of arginine, lysine or any otherphysiologically and pharmaceutically acceptable amine.

A process for the synthesis of the compounds of formula (I) is describedin detail over the course of the present description, making referenceto the synthetic schemes reported in the enclosed FIGS. 1-6.

According to the present invention, compounds of formulae (I), (Ia) and(Ib) may be prepared according to the processes described hereinafter.

Particularly, compounds of general formulae (Ia) 6,5-trans- and (Ib)6,5-cis-fused, wherein p is 1, and n is 1 may be prepared according to asynthetic process outlined in Scheme 1, comprising the following stages:

-   -   a) hydrogenation of the isoxazolidine of compound 1 or of        compound 2, for example with H2, Pd/C in MeOH;    -   b) protection of the amine group with a suitable protective        group, such as for example Cbz, Boc, etc.;    -   c) transformation of the free hydroxyl group into an azide        through the Mitsunobu reaction, or by means of any other known        method (for example, transformation into mesylate and subsequent        nucleophilic substitution with sodium azide), to give compounds        of formulae 6, 9;    -   d) reduction of the azide group into an amino group through the        Staudinger reaction, or by means of hydrogenation to give        compounds 7 and 10.

The compounds of general formulae (Ia) 7,5-trans and (Ib) 7,5-cis fused,wherein p is 1 and n is 2 may be prepared according to a syntheticprocess outlined in Scheme 2, comprising the following stages:

-   -   a) hydrogenation of the isoxazolidine of compound 3 or of        compound 4, for example with H2, Pd/C in MeOH;    -   b) protection of the amine group with a suitable protective        group, such as for example Cbz, Boc, etc.;    -   c) transformation of the hydroxyl group into an azide through        the Mitsunobu reaction, or by means of any other known method        (transformation into mesylate and subsequent nucleophilic        substitution with sodium azide), to give compounds of formulae        12, 15;    -   d) reduction of the azide group into an amino group through the        Staudinger reaction, or by means of hydrogenation to give        compounds 13, 16.

Corresponding compounds wherein X is —N═C═O or —N═S═O may be obtainedfrom the above amino compounds 13 and 16 according to known procedures,i.e., for example, by reaction of the said compounds with phosgene orthiophosgene, respectively.

Compounds wherein X is —SH may be obtained for example by transformationof the hydroxyl group, through the Mitsunobu reaction, with thiolaceticacid (Tetrahedron Lett. 1981, 22, 3119), or by means of any other knownmethods.

Compounds of formula (I) wherein p is from 2 to 5 can be obtainedaccording to known procedures, preferably starting from the abovehydroxyl groups. For example, the compound of formula (I) where p is 2can be obtained from the corresponding compound where p is 1 and X is OHby mesylation, reaction with NaCN and reduction of the nitrile. Inanother example, compound of formula (I) where p is 3 can be obtainedfrom the corresponding compound where p is 1 and X is OH by oxidation toaldehyde, Wittig reaction with diethyl cyanomethylphosphonate andhydrogenation.

The tricyclic starting compounds 1-4 of FIGS. 1 and 2 may be preparedaccording to the procedures described in FIG. 6—Scheme A—Preparation ofthe starting products.

Then, the substitution of the R₁ and R₂ groups in formula (I) with theArg-Gly-Asp (RGD) chain provides compounds which are able are able toselectively bind to integrin receptors and to act as selective inhibitorfor αvβ3 and αvβ5 integrins, of general formula (II)

wherein:

n is 1 or 2,

p is an integer between 1 and 5,

R₄ and R₅ together constitute the sequence Asp-Gly-Arg,

X is —N3, —NHR₃, —SR₃, —N═C═O, or —N═C═S, where:

R₃═H or a protective group;

their salts, racemic mixtures, individual enantiomers, individualdiastereoisomers and mixtures thereof in whatever proportion.

As before, the following configurations may be highlighted for thecompounds of formula (II)

wherein n, R₄, R₅ and X are as defined above and the wedge-shaped anddashed bonds indicate that the substituents are positioned above andbelow the plane respectively.

The peptide sequence Asp-Gly-Arg is advantageously bound to compounds(II), (IIa) and (IIIb) in such a manner whereby the carboxyl group isattached to the amino acid arginine, and the amino group is attached toaspartic acid.

The details provided above for the variable substituents and the saltsof the compounds of formula (I) are also applicable to the compounds offormulae (II), (IIa) and (IIb).

The Asp-Gly-Arg chain may be introduced by adapting the compounds offormulae (I), (Ia) and (Ib) described above, according to a processcomprising the following stages (Scheme 3):

-   -   when R₂ is a protective group, chemoselective deprotection        reaction of the carboxyl group of compound of general        formula (I) and condensation with the appropriately protected        Arg-Gly dipeptide;    -   reduction of the oxazolidine by means of catalytic        hydrogenation;    -   transformation of the methyl ester of glycine into the benzyl        ester through a transesterification reaction, followed by the        simultaneous removal of the protective group from the glycine        and the amino group from the aspartic acid by catalytic        hydrogenation;    -   condensation agent mediated intramolecular cyclisation and        subsequent deprotection of the amino acid side chain protective        groups.

The functional group protection and deprotection reactions may becarried out in accordance with known techniques.

Compounds (IIa) and (IIb) may hence be obtained, according to a processcomprising the following stages (schemes 4-5):

-   -   transformation of the hydroxyl group of compounds 17, 18, 19, 20        into the corresponding azides according to known procedures, for        example through the Mitsunobu reaction, or mesylation and        subsequent nucleophilic substitution with sodium azide, to give        compounds 21, 23, 2, 27;    -   subsequent reduction by means of catalytic hydrogenation or        Staudinger reaction thus providing corresponding amino        derivatives;    -   optional transformation of the amino groups into corresponding        cyanates or thiocyanates by use of phosgene or thiophosgene,        respectively, using known reactions;    -   optional conjugation with molecules of biological interest by        means of known reactions,    -   subsequent deprotection of the aminoacid side chain protective        groups to give the compounds of formulae 22, 24, 23 and 25.

In particular, the preparation of compounds 6,5- and 7,5-cis is reportedin scheme 4 and the preparation of compounds 6,5- and 7,5-trans isreported in scheme 5.

Examples and details of such reactions are provided in the experimentalsection of the present description.

The functional group protection and deprotection reactions may becarried out in accordance with known techniques, such as those describedin the experimental section of the present description.

Details of this kind of preparation for different, even if structurallyanalogous compounds, are provided in WO2005/042531.

The compounds of formula (II), (IIa) and (IIb) thus prepared and bearingany proper X group, as above defined, are further reacted so as to getthe compounds of the invention.

As an example, the compounds of formula (II) wherein X is an optionallyprotected amino group may be reacted with moieties bearing a terminalcarboxy group or a derivative thereof, so as to give rise to acarboxamido linkage.

This reaction, accomplished according to well known operativeconditions, can be schematically represented as follows, for instancefor the preparation of a compound of formula (III) wherein L is a group—CONH—:

R—COOH+H₂N—(CH₂)p-targeting unit---→R₆—CONH—(CH₂)p-targeting unit

Likewise, for instance in the case X is a group —NCO or —NCS, thecompounds of formula (III) wherein L is a group —NHCONH— or —NHCSNH— maybe obtained as follows:

R₆—NH₂+OCN—(CH₂)p-targeting unit---→R₆—NHCONH—(CH₂)p-targeting unit

or

R₆—NH₂+SCN—(CH₂)p-targeting unit---→R₆—NHCSNH—(CH₂)p-targeting unit.

The compounds wherein L is a group (iv) may be obtained by reacting thecorresponding derivative of formula (II) wherein X is azido (—N₃) withthe corresponding moiety properly functionalised through a terminalalkino group —C≡CH, according to well known operative conditions, suchas, for example, the so called “click chemistry” 1,3-dipolarcycloaddition reaction, (see, Kolb, H et al, Angew. Chem. Int. Ed. 2001,40, 2004-20021).

See, as an example:

The compounds wherein L is a group (v) may be obtained by reacting thecorresponding derivative of formula (II) wherein X is SH with thecorresponding moiety properly functionalised with a maleimide, accordingto well known operative conditions (see Brinkley, M. Bioconjugate Chem.1992, 3, 2-13).

See, as an example:

Clearly, the above schemes may apply as well for the preparation of thecompounds of formula (III) of the invention wherein L is a divalentlinking moiety comprising any one of the aforementioned (i) to (v)groups.

In such a case, the following order of reactions may apply, for instancein the preparation of a compound of formula (III), wherein L is thefollowing group:

—NHOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—CONH—

R₆—NH₂+HOOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—COOH→

→R₆—HNOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—COOH and, thenR₆—HNOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—COOH+H₂N—(CH₂)p-targeting unit→R₆—HNOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—CONH—(CH₂)p-targeting unit.

From the above it is clear to the skilled person that, wheneverconvenient, the order of the reactions may be properly varied,substantially as follows:

HOOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—COOH+H₂N—(CH₂)p-targeting unit→

→HOOC—CH₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₂—CONH—(CH₂)p-targeting unit and, then+R₆—NH₂ so as to get the compound of formula (III).

Any of the said cross-linking or coupling reactions may be performedaccording to appropriate reactions well known to those skilled in theart.

Thus, for examples, the lactamic structures including an azido group maybe bound the desired biologically active molecules or compounds ofbiological importance through appropriate reactions i.e., for example,Click chemistry.

When the lactamic structures includes an amine group, the biologicallyactive moiety may be bound, for example, through a simple amidationreaction.

Moreover, the cyclic peptidomimetic wherein X═—NH—CO or —NH—CS may bebound to suitable amine group of the biologically active moleculeaccording to procedures known to the person skilled in the art to give,for example, corresponding ureas or thioureas derivatives.

Examples and details concerning the former functional groups andcross-linking reaction between them are provided in the experimentalsection of the present description.

Preferably, in case L is other than a group (i) to (v) within thecompounds of the invention, it may be conveniently derived from thefollowing molecules (shortly referred to as linkers) listed in table Ibelow

TABLE 1 LINKER A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

A21

A22

A23

A24

A25

A26

A27

A28

A29

A30

A31

or it may be even obtained from a suitable combination thereof thusproviding modified and/or elongated compounds such as, for example,

Most of the linkers of Table 1 are well known and commercially availablefrom, for example, Aldrich Neosystem and Peptides Internationalcatalogs. The non-marketed compounds may be easily prepared according toknown methods, for example as per the accompanying bibliographicreferences.

The compound A 32 is new and it constitutes a further object of thepresent invention.

As formerly explained, the above linkers or suitable combinationsthereof may be connected to X, from one side, and to R₆, from the otherside, by use of cross-linking or coupling reactions well known in theart. When X is an azide group, for example, one approach, may be basedon the so called “click chemistry” 1,3-dipolar cycloaddition reaction,(see, f.e., Kolb, H et al, Angew. Chem. Int. Ed. 2001, 40, 2004-20021),between the said azide group and a terminal acetilenic group of thebifunctional moiety. This kind of approach is best detailed in theexperimental section below. This same kind of reaction, obviously allowsto connect an acetylenic group on the peptide side with a linker bearinga terminal azido group.

When X is SH—, a suitable reaction may occur with a bifunctional linkerincluding, for example, a maleimido terminal group such as, for instancethose listed as A22-A24 in Table 1.

When X is NH₂, the linker may suitably include a C terminus, i.e. acarboxylic terminal group. The connection between L and R₆ may bethrough a number of arrangements, including, e.g.,: (a) from C-terminusto C-terminus; (b) from N-terminus(i.e. a terminal amine group) toC-terminus; (c) from C-terminus to N-terminus; or (d) from N-terminus toN-terminus depending on the binding groups of L and R₆ involved in thecross-linking reaction.

The above coupling reactions may further apply when considering thepreparation of all of the compounds of the invention deriving fromcoupling one or more peptidomimetic moieties of formula (II), ananchoring system selected from those of FIG. 7A-7C, one or more R₆biologically active moieties of the invention, through one or morelinking units.

Obviously, when operating all the above coupling reactions, the optionalfunctional groups on the coupling molecules that are not involved in thesaid reactions must be protected to avoid undesired bonds being formed.The protecting groups that can be used are listed, for example, inGreene, Protective groups in Organic Synthesis, John Wiley & Sons, NewYork (1981).

Chelates

In the case of R₆, as per the compounds of the invention, is adiagnostic or radiotherapeutic agent, for instance a metal chelate or asystem containing several or many metal chelates of any suitable metalion among those formerly reported, complexation may occurs by properlylabelling any compound of the invention wherein R₆ is or comprises achelating unit or units with the proper metal of choice, according towell known methods.

For example, the paramagnetic complexes of the invention and,particularly, the Gd(III) chelates may be prepared by stoichiometricaddition of suitable Gd(III) derivatives, particularly Gd(III) salts oroxides. See, for instance, EP 230893 disclosing labelling withparamagnetic metal ions, and WO 98/52618, U.S. Pat. No. 5,879,658, andU.S. Pat. No. 5,849,261 disclosing labelling with radioactive metals.

As an example, complexes of radioactive technetium or indium of theinvention are particularly useful for diagnostic imaging whilstcomplexes of radioactive rhenium are particularly useful forradiotherapy.

When considering PET imaging, complexes of Ga-68 and Cu-64 as well asvarious Lanthanides are particularly useful.

In forming a complex of radioactive technetium, for instance, atechnetium complex, preferably a salt of ^(99m)Tc pertechnetate, isreacted with the unlabelled compounds of the invention in the presenceof a reducing agent. Preferred reducing agents are dithionite, stannousand ferrous ions; the most preferred reducing agent is stannouschloride. Means for preparing such complexes are conveniently providedin a kit form comprising a sealed vial containing a predeterminedquantity of compound of the invention to be labeled and a sufficientamount of reducing agent to label the reagent with ^(99m)Tc.Alternatively, the complex may be formed by reacting a compounds of thisinvention, which is conjugated with an appropriate chelating moiety witha pre-formed labile complex of technetium and another compound known asa transfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the ^(99m)Tc pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts. Preparation ofthe complexes of the present invention where the metal is radioactiverhenium may be accomplished using as starting materials rheniumcompounds wherein the metal is in the +5 or +7 oxidation state. Examplesof compounds in which rhenium is in the Re(VII) state are NH₄ReO₄ orKReO₄. Re(V) is available as, for example, [ReOCl₄](NBu₄),[ReOC₄](AsPh₄), ReOCl₃(PPh₃)₂ and as ReO₂(pyridine)₄ ₊ (Ph is phenyl; Buis n-butyl). Other rhenium reagents capable of forming a rhenium complexmay also be used.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention must contain a suitable amount of radioactivity. Informing ¹¹¹In or ^(99m)Tc complexes, it is generally preferred to formradioactive complexes in solutions containing radioactivity atconcentrations of from about 0.01 millicurie (mCi) to 100 mCi per mL.

The compounds of the invention find a variety of applications in thetherapeutic and diagnostic field.

MRI contrast agents according to the present invention, for instance,may be used in the same manner as conventional MRI contrast reagents.When the target is, for example, an angiogenic site in a tissue, certainMR techniques and pulse sequences may be preferred to enhance thecontrast of the site to the background blood and tissues. Thesetechniques include (but are not limited to), for example, black bloodangiography sequences that seek to make blood dark, such as fast spinecho sequences (see, e.g., Alexander et al., Magnetic Resonance inMedicine, 40(2): 298-310 (1998)) and flow-spoiled gradient echosequences (see, e.g., Edelman et al., Radiology, 177(1): 45-50 (1990)).These methods also include flow independent techniques that enhance thedifference in contrast, such as inversion-recovery prepared orsaturation-recovery prepared sequences that will increase the contrastbetween target containing tissue, such as an angiogenic tumor, andbackground tissues. Finally, magnetization transfer preparations mayalso improve contrast with these agents (see, e.g., Goodrich et al.,Investigative Radiology, 31(6): 323-32 (1996)).

The paramagnetic contrast agents of the invention, for use in MRItechniques, are administered to the patient in the form of an injectablecompositions. The method of administering the MRI contrast agent ispreferably parenterally, meaning intravenously, intraarterially,intrathecally, interstitially, or intracavitarilly. For imaging activeangiogenesis, intravenous or intraarterial administration is preferred.

For MRI, it is contemplated that the subject will receive a dosage ofcontrast agent sufficient to enhance the MR signal at the target (e.g.,a site of angiogenesis) at least 10%. After injection of the targetedcontrast agent of the invention including the MRI agent, the patient isscanned in the MRI machine to determine the location of any sitescontaining the target. In therapeutic settings, upon targetlocalization, a cytotoxic or therapeutic agent can be immediatelyadministered, if necessary, and the patient can be subsequently scannedto visualize the therapeutic effect.

In case of radiotherapy, proper dose schedules known in the art may beused for the radiotherapeutic compounds of the present invention.

The compounds can be administered using many methods which include, butare not limited to, a single or multiple IV or IP injections, using aquantity of radioactivity that is sufficient to cause damage or ablationof the targeted tissue, but not so much that substantive damage iscaused to non-target (normal tissue). The quantity and dose required isdifferent for different constructs, depending on the energy andhalf-life of the isotope that is used, the degree of uptake andclearance of the agent from the body and the mass of the tumor. Ingeneral, doses can range from about 0.01 mCi to about 100 mCi,preferably from 1 mCi to 50 mCi. Typically, a single dose of about 30-50mCi to a cumulative dose of up to about 3 Curies may apply.

The radiotherapeutic compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and may include, for example, para-aminobenzoic acid, ascorbic acid,gentisic acid and the like.

In case of radionuclide imaging, the compound of the invention may beadministered to the patient through injection. A PET camera or a gammacamera calibrated for the gamma ray energy of the nuclide incorporatedin the imaging agent is used to image areas of uptake of the agent andquantify the amount of radioactivity present in the site. Imaging of thesite in vivo can take place in a matter of a few minutes. However,imaging can take place, if desired, in hours or even longer, after theradiolabeled peptide is injected into a patient. In most instances, asufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 of an hour to permit the taking ofscintiphotos.

A single, or multi-vial kit that contains all of the components neededto prepare the radiopharmaceuticals of this invention, other than theradionuclide, is an integral part of this invention.

A single-vial kit preferably contains a chelating ligand (if a metalradionuclide is used), a source of a stannous salt (if reduction isrequired, e.g., when using technetium), or other pharmaceuticallyacceptable reducing agent, and is appropriately buffered withpharmaceutically acceptable acid or base to adjust the pH to a value ofabout 3 to about 9. The quantity and type of reducing agent used woulddepend highly on the nature of the exchange complex to be formed. Theproper conditions are well known to those that are skilled in the art.It is preferred that the kit contents be in lyophilized form. Such asingle vial kit may optionally contain labile or exchange ligands suchas glucoheptonate, gluconate, mannitol, malate, citric or tartaric acidand can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or α-, β-, or γ-cyciodextrin that serve to improve theradiochemical purity and stability of the final product. The kit mayalso contain stabilizers, bulking agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial may contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g., the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains theligand, as well as buffers appropriate to adjust the pH to its optimalvalue. After a reaction time of about 5 to 60 minutes, the complexes ofthe present invention are formed. It is advantageous that the contentsof both vials of this multi-vial kit be lyophilized. As above, reactionmodifiers, exchange ligands, stabilizers, bulking agents, etc. may bepresent in either or both vials.

As above reported, the compounds of the invention can be suitablyformulated according to known methods and the compositions thereof dorepresent an additional object of the invention.

In order to obtain the desired prophylactic, therapeutic or diagnosticeffect, a therapeutically or diagnostically effective dose or amount ofthe active ingredient is advantageously administered in the form of aunit dose, one or more times daily. The daily dosages are obviouslyselected by the health professional depending on the biologically activemolecule introduced.

The term “effective dose or amount”, as used herein, refers to anyamount of a diagnostic or a therapeutic molecule of the invention, orpharmaceutical composition thereof, that is sufficient to fulfil itsintended diagnostic or therapeutic purpose(s): i.e., for example, tovisualize a patient biological element including cells, biologicalfluids and biological tissues as well as human body organs, regions ortissues affected by angiogenesis, or its intended therapeuticpurpose(s); or to delay or to prevent to onset of a pathologicalcondition associated with angiogenesis; or to slow down or stop theprogression, aggravation, or deterioration of the symptoms.

The diagnostic or therapeutic agents of the invention have a wide rangeof applications as they can be used for oral, intravasal, (for instanceintravenous, intraarterial, intracoronaric, intraventricularadministration and the like), intrathecal, intraperitoneal,intralymphatic and intracavital administrations. Compositions for thedesired route of administration can be prepared by any of the methodswell known in the pharmaceutical arts. Details concerning dosages,dosage forms, modes of administration, composition and the like arefurther discussed in a standard pharmaceutical text, such as Remington'sPharmaceutical Sciences, 18th Ed., Alfonso R. Gennaro, ed. (MackPublishing Co., Easton, Pa. 1990), which is hereby incorporated byreference.

In one preferred embodiment, a suitable parmaceutical compositionaccording to the invention is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenousadministration to human being. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Otherpharmaceutically acceptable carriers include, but are not limited to,sterile water, saline solution, buffered saline water, saline solution,buffered saline (including buffers like phosphate or acetate), alcohol,vegetable oils, polyethylene glycols, gelatin, lactose, amylose,magnesium stearate, talc, silicic acid, paraffin, etc. Where necessary,the composition may also include a solubilizing agent and a localanaesthetic such as, e.g., lidocaine to ease pain at the site of theinjection; and further may include preservatives, stabilizers, wettingagents, emulsifiers, salts, lubricants, etc. as long as they do notreact deleteriously with the active compounds. Similarly, thecomposition may comprise conventional excipients, i.e., pharmaceuticallyacceptable organic or inorganic carrier substances suitable forparenteral, enteral or intranasal application which do not deleteriouslyreact with the active compounds. Generally, the ingredients will besupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as, e.g., an ampoule or sachetteindicating the quantity of active agent in activity units. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade “water forinjection” or saline. Where the composition is to be administered byinjection, an ampoule of sterile water for injection or saline may beprovided so that the ingredients may be mixed prior to administration.

Such pharmaceutical compositions are preferably formulated forparenteral administration, and most preferably for intravenous orintra-arterial administration. Generally, and particularly whenadministration is intravenous or intra-arterial, pharmaceuticalcompositions may be given as a bolus, as two or more doses separated intime, or as a constant or non-linear flow infusion.

The diagnostic or therapeutic agents of the invention can beadministered to an individual over a suitable time course, depending onthe nature of the condition and the desired outcome. As describedherein, the compounds of the present invention can further beadministered systemically or locally including, for example, throughtopical application, transdermal, parenteral, gastrointestinal,intravaginal, and transalveolar administration.

For topical applications, the compounds of the invention can besuspended, for example, in a cream, gel or rinse which allows thepeptidic derivatives or multimeric constructs to penetrate the skin andenter the blood stream, for systemic delivery, or contact the are ofinterest, for localized delivery. Compositions suitable for topicalapplication include any pharmaceutically acceptable base in which thepolypeptides are at least minimally soluble. For transdermaladministration, the compounds of the invention can be applied inpharmaceutically acceptable suspension together with a suitabletransdermal device or “patch.” Examples of suitable transdermal devicesfor administration of the diagnostic or therapeutic agents of thepresent invention are described, for example, in U.S. Pat. No.6,165,458, issued Dec. 26, 2000 to Foldvari, et al., and U.S. Pat. No.6,274,166B1, issued Aug. 4, 2001 to Sintov, et ai., the teachings ofwhich are incorporated herein by reference.

For parenteral administration, the diagnostic or therapeutic agents ofthe invention can be suspended, for example, in a pharmaceuticallyacceptable sterile isotonic solution, such as saline and phosphatebuffered saline. Then they may be injected intravenously,intramuscularly, intraperitoneally, or subcutaneously.

For the oral administration, the agents of the invention can beformulated according to preparation methods routinely used in thepharmaceutical technique or as coated formulations to gain additionalprotection against the stomach acidic pH, thus preventing the chelatedmetal ion from release, which takes place particularly at the typical pHvalues of gastric fluids.

Other excipients, for example including sweeteners and/or flavouringagents, can also be added, according to known techniques ofpharmaceutical formulations.

For gastrointestinal and intravaginal administration, the compounds ofthe invention can be incorporated into pharmaceutically acceptablepowders, pills or liquids for ingestion, and suppositories for rectal orvaginal administration.

For transalveolar, buccal or pulmonary administration, the diagnostic ortherapeutic agents pf the invention can be suspended in apharmaceutically acceptable excipient suitable for aerosolization andinhalation or as a mouthwash. Devices suitable for transalveolaradministration such as atomizers and vaporizes are also included withinthe scope of the invention. Suitable formulations for aerosol deliveryof polypeptides using buccal or pulmonary routes can be found, forexample in U.S. Pat. No. 6,312,665B1, issued Nov. 6, 2001 to PankajModi, the teachings of which are incorporated herein by reference.

In addition, the agents of the invention can be administered nasally orocularly, where the diagnostic or therapeutic compounds of the inventionare suspended in a liquid pharmaceutically acceptable agent suitable fordropwise dosing.

In an even further aspect the invention relates to the use of the saidnovel diagnostic agents for the preparation of a diagnostic formulationfor use in the diagnostic imaging, both in vitro and in vivo, ofpathological systems, including cells, biological fluids and biologicaltissues originating from a live mammal patient, and preferably, humanpatient, as well as of human body organ, regions or tissues affected byangiogenic processes, including tumorous or cancerous tissues,inflammations, over-expressing integrin and particularly αvβ3 integrinreceptors, as well as for monitoring the progress and results oftherapeutic treatment of the said pathologies. In yet another aspect theinvention provides a method for imaging angiogenesis both in vitro andin vivo comprising the use of a diagnostic imaging agent of theinvention targeted to integrin receptors and an imaging technique.

In a preferred aspect the invention provides a method of in vivo imagingof a patient comprising: administering, by injection or infusion, to apatient an imaging effective amount of a diagnostic agent of formulae(III), (VI), (VII) and (VIII) wherein the peptidic moiety of the saiddiagnostic compound allows the imaging agent to interact with integrinsso consenting thereof detection by use of an imaging technique.

In a further aspect, the invention concerns a therapeutic agentaccording to anyone of formulae (III), (VI) or (VII) wherein R₆ is atherapeutically effective moiety and, preferably, a radiotherapeuticalagent, or in the form of therapeutically effective macromolecularaggregate comprising on their surface a number of targeting moietiesaccording to the invention.

In still further aspect the invention provides a method of preventing orinhibiting angiogenesis both in vitro and in vivo comprising contactinga pathological systems, including cells, biological fluids, biologicaltissues, originating from a live mammal patient, and preferably, humanpatient, or a body organ, tissue or area exhibiting angiogenicvasculature with a therapeutic agent of the invention.

EXPERIMENTAL SECTION Terms and Abbreviations Definition

-   CEST Contrast Enhanced Saturation Transfer-   ATCC: American Type Culture Cell-   MEM: Minimum Essential Medium-   O.G.: Official Gazette-   PBS: Dulbecco's Phosphate Buffered Saline without Ca++ and Mg++-   RT: Room Temperature-   EBM: Endothelial Cell Basal Medium-   EGM: Endothelial Growth Medium-   HUVEC: Human Umbilical Vein Endothelial Cell-   DMEM: Dulbecco's Modified Eagle's Medium-   FCS: Foetal Calf Serum-   RGD: Arg-Gly-Asp-   Fmoc or fmoc 9-fluorenylmethyloxycarbonyl-   NHS N-hydroxysuccinimide-   DMF dimethylformamide-   DMAP 4-dimethylaminopyridine-   TEA Triethylamine-   MeCN acetonitrile-   MsCl methanesulfonylchloride-   DCM dichloromethane-   DMSO dimethyl sulfoxide-   TFA trifluoroacetic acid-   Cbz Carbobenzyloxy-   Boc t-butyloxycarbonyl-   Ser Serine-   Pro Proline-   Gly Glycine-   Arg Arginine-   POPC 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine.-   DSPE-PEG2000Biotin    1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Biotinyi(Poyethylene    Glycol)2000](Ammonium Salt)-   DPDP 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine-   DPPC 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine-   DIPEA Diisopropylethylamine-   HBTU (2-(1H-Benzotriazol-1-yl)-1,2,3,3-tetramethyluronium    hexafluorophosphate)-   ST Saturation Transfer

Preparation and tests concerning novel cyclic pentapeptide derivativesof the invention are illustrated in the following examples. The specificparameters included in the following examples are intended to illustratethe practice of the invention, and they are not presented to in any waylimit the scope of the invention. A skilled technician may understandthat different preparation approaches may equally be adopted based onsynthetic procedures well known in the art.

General Observations:

The reported ¹H-NMR and ¹³C-NMR data for prepared compounds have beenrecorded in the solvents indicated using a Briiker Avance-400 instrumentat 400 MHz and 100.6 MHz respectively. Chemical shift values areindicated in ppm and the coupling constants in Hz. Optical rotatorypowers are measured using a Perkin-Elmer model 241 polarimeter. Thinlayer chromatography (TLC) is performed using Merck F-254 plates. Flashchromatography is performed using Macherey-Nagel 60, 230-400 mesh silicagel. Solvents are anhydrified in accordance with standard procedures andreactions requiring anhydrous conditions are carried out in an argonatmosphere. FAB⁺ mass spectrometry has been performed using a VG 7070EQ-HF spectrophotometer, ESI⁺ mass spectrometry has been performed usinga Bruker Esquire 3000 plus spectrophotometer.

LIPOCEST agents have been characterised by ¹H-NMR using a Bruker Avance600 spectrometer. Details on each of the adopted conditions are cited inthe examples below.

The MR-CEST images have been acquired on a Bruker Avance 300spectrometer equipped with a microimaging probe.

Preparation of the Cyclic Pentapeptides

Functionalization of the Compounds Deriving 1,3 dipolar Cyclization(Scheme 1 and Scheme 2).

a) Protection of the Free Amino Group

To a solution of product 5 or 14 (0.51 mmol) in anhydrous CH₂Cl₂ (5 ml)under argon atmosphere and at room temp., are added in the followingsequence TEA (184 μl, 1.33 mmol), Cbz-Cl (95 μl, 0.61 mmol) and finallyDMAP (15 mg, 0.126 mmol). The solution is kept under stirring forapprox. 18 hours. After this period of time, it is taken up with CH₂Cl₂(5 ml) and washed with NH₄Cl (2×5 ml). The organic phase, dried overNa₂SO₄, is taken to evaporated and the crude product thus obtained ispurified by flash chromatography (EtOAc/ETP 7:3→8:2) to give the desiredproduct as a white foam (60%-78%).

Characterization of Compound 5-Cbz

Yield: 78%. [α]_(D) ²²=−13.7 (c=1.0, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ1.48 (s, 9H, C(CH₃)₃), 1.6 (m, 1H, H-5), 1.7 (m, 1H, H-7), 2.06 (m, 1H,H-8), 2.2 (m, 1H, H-8), 2.23 (m, 1H, H-7), 2.3 (m, 1H, H-5), 2.75 (m,1H, Ht-4), 2.92 (bs, 1H, OH), 3.6 (dd, 1H, HCHOH), 3.71 (m, 1H, HCHOH),3.72 (m, 1H, H-6), 4.38 (t, 1H, H-3), 4.48 (d, 1H, H-9), 5.15 (dd, 2H,CH₂Ph), 6.0 (d, 1H, NHCbz), 7.28-7.42 (m, 5H, aromatic protons). ¹³C NMR(100.6 MHz, CDCl₃): δ 171.7, 168.1, 156.7, 136.4, 128.5, 128.8, 127.9,82.4, 66.9, 63.5, 58.9, 55.7, 52.1, 37.9, 32.5, 32.0, 31.6, 29.7, 29.2,27.9. MS [FAB⁺]: 419.3 [M+1]⁺.

Calculated elemental analysis C₂₂H₃₀N₂O₆: C, 63.14; H, 7.23; N, 6.69;observed C, 62.16; H, 7.25; N, 6.67.

Characterization of Compound 14-Cbz

Yield: 60%. [α]_(D) ²²=−25.5 (c=1.0, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ1.48 (s, 9H, C(CH₃)₃), 1.51 (m, 1H, H-6), 1.53 (m, 1H, H-4), 1.75 (m,1H, H-5), 1.83 (m, 1H, H-6), 1.98 (m, 1H, H-9), 2.0 (m, 1H, H-5), 2.2(m, 1-t, H-9), 2.25 (m, 1H, H-8), 2.33 (m, 1H, H-8), 3.4 (t, 1H, HCHOH),3.72 (d, 1H, OH), 3.82 (d, 1H, HCHOH), 4.03 (t, 1H, H-7), 4.42 (dd, 1H,H-3), 4.5 (d, 1H, H-10), 5.15 (dd, 2H, CH₂Ph), 6.48 (d, 1H, NHCbz),7.31-7.43 (m, 5H, aromatic protons). ¹³C NMR (100.6 MHz, CDCl₃): δ170.5, 170.3, 158.0, 136.0, 128.6, 128.2, 128.1, 81.6, 67.5, 64.5, 61.0,58.7, 55.5, 42.5, 33.9, 32.1, 30.9, 29.7, 29.3, 28.0, 27.2. MS [ESI⁺]:433.3 [M+H]⁺, 455.3 [M+Na]⁺, Calculated elemental analysis C₂₃H₃₂N₂O₆:C, 63.87; H, 7.46; N, 6.48; observed C, 63.85; H, 7.47; N, 6.47.

b) Synthesis of Azide-Derivatives

To a solution of product 5-Cbz or 14-Cbz (0.29 mmol) in anhydrous CH₂Cl(4 ml) under argon atmosphere and at room temp., are added in thefollowing sequence MsCl (846 μl, 0.59 mmol) and TEA (165 ti, 1.18 mmol).The solution is kept under stirring for approx. 45 minutes. After thisperiod of time, it is taken up with CH₂Cl and washed with NH₄Cl. Theorganic phase, dried over Na₂SO₄ is evaporated and the crude productthus obtained dissolved in DMF (3.2 ml) and, under argon atmosphere andat room temp., NaN₃ (154 mg, 2.37 mmol) is added. The reaction is keptunder stirring at 80° C. for approx. 18 hours. After this period oftime, the DMF is evaporated off to dryness, and the crude productdissolved in CH₂Cl₂ and washed with H₂O. The organic phase, dried overNa₂SO₄, is taken to dryness and the crude product thus obtained ispurified by flash chromatography (AcOEt/ETP 7:3) to give the desiredproduct as a white foam (76%-90%).

Characterization of Compound 6

Yield: 76%. [α]_(D) ²²=+19.0 (c=1.0, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ1.48 (s, 9H, C(CH₃)₃), 1.61 (m, 1H, H-5), 1.72 (m, 1H, H-7), 2.08 (m,1H, H-8), 2.14 (m, 1H, H-8), 2.24 (m, 1H, H-7), 2.31 (m, 1H, H-5), 2.9(m, 1H, H-4), 3.28 (dd, 1H, J=Hz, HCHN₃), 3.48 (dd, 1H, HCHN₃), 3.7 (m,1H, H-6), 4.31 (t, 1H, H-3), 4.39 (d, 1H, H-9), 5.15 (s, 2H, CH₂Ph), 6.0(bs, 1H, NHCbz), 7.28-7.42 (m, 5H, aromatic protons). ¹³C NMR (100.6MHz, CDCl₃): δ 170.4, 166.7, 156.1, 136.3, 128.5, 128.1, 127.9, 81.9,67.0, 59.0, 55.3, 55.8, 52.6, 35.4, 32.3, 31.8, 29.2, 28.0. MS [ESI⁺]:444.3 [M+H]⁺. Calculated elemental analysis C₂₂H₂₉N₅O₅: C, 59.58; H,6.59; N, 15.79; observed C, 59.57; H, 6.58; N, 15.81.

Characterization of Compound 15

Yield: 90%. [α]_(D) ²²=−13.8 (c=1.0, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ1.48 (s, 9H, C(CH₃)₃), 1.5 (m, 1H, H-5), 1.68 (m, 1H, H-4), 1.70 (m, 1H,H-6), 1.72 (m, 1H, H8), 1.82 (m, 1H, H-5), 1.98 (m, 1H, H-9), 2.13 (m,1H, H-6), 2.21 (m, 1H, H-9), 2.34 (m, 1H, H-8), 3.26 (dd, 1H, J=18.9 Hz,J=12.1 Hz, HCHN₃), 3.65 (dd, 1H, HCHN₃), 4.09 (t, 1H, J=9.0 Hz, H-7),4.42 (dd, 1H, H-3), 4.46 (dd, 1H, J=8.7 Hz, J=2.0 Hz, H-10), 5.12 (dd,2H, J=15.7 Hz, J=12.2 Hz, CH₂Ph), 6.08 (d, 1H, J=7.18 Hz, NHCbz),7.3-7.42 (m, 5H, aromatic protons). ¹³C NMR (100.6 MHz, CDCl₃): δ 170.5,169.7, 156.6, 136.3, 128.5, 128.1, 81.6, 67.1, 60.9, 58.3, 55.9, 53.7,40.9, 33.3, 31.9, 31.5, 28.0, 27.2. MS [ESI+]: 448.2 [M+H]⁺, 480.2[M+Na]⁺, Calculated elemental analysis C₂₃H₃₁N₅O₅: C, 60.38; H, 6.83; N,15.31; observed C, 60.36; H, 6.84; N, 15,32.

c) Reduction of Azide-Derivatives

To a solution of product 6 or 15 (0.034 mmol) in anhydrous CH₂Cl₂ (350μl) under argon atmosphere and at room temp., is added 1M Me₃P intoluene (51 μl, 0.051 mmol). After approx. 2 hours, upon completion ofthe reaction, the reaction is taken up with CH₂Cl₂ (1 ml), and H₂O (1ml) added, and the mixture is allowed under stirring for approx. 10minutes. After this period of time, the two phases are separated. Theorganic phase, dried over Na₂SO₄, is evaporated to dryness.

Characterization of Compound 7

Yield: 93%. [α]_(D) ²²=−13.9 (c=1.0, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ1.47 (s, 9H, C(CH₃)₃), 1.55 (m, 1H, H-5), 1.72 (m, 1H, H-7), 2.07 (m,1H, H-8), 2.16 (m, 1H, H-8), 2.21 (m, 1H, H-7), 2.30 (m, 1H, H-5), 2.68(m, 1H, H-4), 2.72-2.97 (m, 2H, CH₂NH₂), 3.73 (m, 1H, H-6), 4.31 (t, 1H,H-3), 4.41 (d, 1H, H-9), 5.15 (s, 2H, CH₂Ph), 6.4 (d, 1H, NHCbz),7.23-7.42 (m, 5H, aromatic protons). ¹³C NMR (100.6 MHz, CDCl₃): δ170.8, 168.1, 156.4, 136.6, 128.5, 128.0, 127.9, 81.8, 67.8, 58.9, 55.6,53.0, 32.5, 32.0, 29.7, 29.3, 28.0. MS [ESI⁺]: 418.4 [M+H]⁺. Calculatedelemental analysis C₂₂H₃₁N₃O₅: C, 63.29; H, 7.48; N, 10.06; observed C,63.27; H, 7.47; N, 10.08.

Characterization of Compound 16

Yield: 76%. [α]_(D) ²²=−14.5 (c=1.0, CHCl₃). ¹H-NMR (400 MHz, CDCl₃): δ1.45 (s, 9H, C(CH₃)₃), 1.5 (m, 1H, H-5), 1.53 (m, 1H, H-4), 1.65 (m, 1H,H-6), 1.72 (m, 1H, H8), 1.81 (m, 1H, H-5), 1.98 (m, 1H, H-9), 2.14 (m,1H, H-6), 2.21 (m, 1H, H-9), 2.33 (m, 1H, H-8), 2.83 (bs, 2H, CH₂NH₂),4.08 (t, 1H, J=9.0 Hz, H-7), 4.41 (m, 1H, H-3), 4.47 (d, 1H, J=8.5 Hz,H-10), 5.13 (dd, 2H, J=15.7 Hz, J=12.2 Hz, CH₂Ph), 6.16 (d, 1H, J=7.0Hz, NHCbz), 7.28-7.42 (m, 5H, aromatic protons). ¹³C NMR (100.6 MHz,CDCl₃): δ 170.6, 156.8, 136.2, 128.5, 128.0, 81.5, 67.0, 60.8, 58.3,56.3, 44.4, 33.7, 32.0, 31.3, 28.0, 27.2. MS [ESI⁺]: 432.5 [M+H]⁺.Calculated elemental analysis C₂₃H₃₃N₃O₅: C, 64.02, H, 7.71, N, 9.74;observed C, 64.04; H, 7.72; N, 9.73

Preparation of Amide Derivatives of the Cyclic Pentapeptide Containingthe RGD Sequence (Scheme 4 and Scheme 5)

a) Synthesis of azide-derivatives

To a solution of product 17-20 (0.055 mmol) in anhydrous CH₂Cl₂ (700 μl)under argon atmosphere and at room temp., are added in the followingsequence MsCl (8.5 μl, 0.11 mmol) and TEA (30 l, 0.22 mmol). Thesolution is kept under stirring for approx. 30 minutes. After thisperiod of time, the solvent is evaporated to dryness and the crudeproduct filtered over silica gel (CH₂Cl₂/MeOH 9:1). The crude productthus obtained is dissolved in DMF (55 μl) and, under argon atmosphereand at room temp., NaN₃ (36 mg, 0.55 mmol) is added. The reaction iskept under stirring at 80° C. for approx. 18 hours. After this period oftime, the DMF is evaporated off to dryness, and the crude productdissolved in CH₂Cl₂ and washed with H₂O. The organic phase, dried overNa₂SO₄, is evaporated and the crude product thus obtained is purified byflash chromatography (CH₂Cl₂/iPrOH 9:1→8:2) to give the desired productas a white foam (30%-75%).

Characterization of Compound 21

Yield: 62%. (White solid). [α]_(D) ²²=−6.3 (c=1.0, CHCl₃). ¹H NMR (400MHz, Acetone-D6): δ 1.23 (s, 6H, C(CH₃)₂ Pmc), 1.38 (s, 9H, C(CH₃)₃),1.44 (m, 1H, Hβ Arg), 1.47 (m, 1H, H-7), 1.49 (m, 1-1H, H-8), 1.51 (m,1H, Hβ Arg), 1.60 (m, 1H, H© Arg), 1.72 (m, 2H, CH₂CH₂Ar Pmc), 1.74 (m,1H, H-5), 1.80 (m, 1H, Hγ Arg), 2.02 (s, 3H, CH₃ Pmc), 2.1 74 (m, 1H,H-5), 2.12 (m, 1H, H-4), 2.18 (m, 1H, H-7), 2.3 (m, 1H, H-8), 2.48 (s,3H, CH₃ Pmc), 2.50 (s, 3H, CH₃ Pmc), 2.54 (m, 2H, CH₂CH₂Ar Pmc), 2.60(m, 1H, Hβ Asp), 2.91 (m, 1H, Hβ Asp), 3.17 (m, 1H, Hδ Arg), 3.19 (m,1H, CH₂N3), 3.23 (m, 1H, Hδ Arg), 3.48 (m, 1H, CH₂N3), 3.6 (m, 1H, HαGly), 3.8 (m, 1H, Hα Gly), 4.0 (m, 1H, H-6), 4.17 (m, 1H, H-9), 4.4 (m,1H, H-3), 4.63 (m, 1H, Hα Arg), 4.72 (m, 1H, Hα Asp), 6.14 (bs, 1H,(NH)₂C═NH), 6.37 (bs, 2H, (NH)₂C═NH), 7.32-7.48 (m, 2H, NH Arg, NHbicyclic), 7.8 (bs, 1H, NH Gly), 8.12 (bs, 1H, NH Asp). ¹³C NMR HETCOR(400 MHz, Acetone-D6): δ 67.5, 54.8, 51.8, 51.4, 50.2, 49.4, 45.8, 40.1,39.9, 34.7, 32.8, 31.8, 31.5, 30.2, 28.1, 27.2, 26.8, 25.2, 21.3, 18.5,17.5, 12.1. MS [FAB⁺]: calculated for C₄₀H₅₉N₁₁O₁₀S: 885.42, observed:886 [M+H]⁺. Calculated analysis for C₄₀H₅₉N₁₁O₁₀S: C, 54.22, H, 6.71, N,17.39; observed C, 54.20; H, 6.72; N, 17.37.

Characterization of Compound 23

Yield: 30%. (White solid). [α]_(D) ²²=−65.15 (c=1.0, Acetone). ¹H NMR(400 MHz, Acetone-D6): δ 1.18 (s, 6H, C(CH₃)₂ Pmc), 1.3 (s, 9H,C(CH₃)₃), 1.44 (m, 1H, H-8), 1.45 (m, 3H, H-6, Hγ Arg), 1.5 (m, 1H, HβArg), 1.57 (m, 1H, Hβ Arg), 1.62 (m, 1H, H-4), 1.65 (m, 1H, H-5), 1.7(m, 2H, CH₂CH₂Ar Pmc), 1.73 (m, 2H, H-5, H-9), 1.95 (m, 1H, H-9), 1.96(s, 3H, CH₃ Pmc), 1.98 (m, 1H, H-6), 2.13 (m, 1H, H-8), 2.46 (s, 3H, CH₃Pmc), 2.48 (s, 3H, CH₃ Pmc), 2.55 (m, 2H, CH₂CH₂Ar Pmc), 2.7 (m, 1H, HβAsp), 2.78 (m, 1H, Hβ Asp), 3.07 (m, 1H, HCHN₃), 3.11 (m, 2H, H6 Arg),3.56 (m, 1H, HCHN₃), 3.63 (m, 1H, Hα Gly), 3.87 (m, 1H, H-7), 3.93 (m,1H, Hα Gly), 4.33 (m, 1H, Hα Asp), 4.35 (m, 1H, H-3), 4.42 (m, 1H, HαArg), 4.64 (m, 1H, H-10), 6.25 (bs, 1H, (NH)₂C═NH), 6.36 (bs, 2H,(NH)₂C═NH), 7.46 (m, 1H, NH bicyclic), 7.77-7.9 (m, 2H, NH Gly, NH Arg),8.05 (bs, 1H, NH Asp). ¹³C NMR HETCOR (400 MHz, Acetone-D6): δ 61.2,58.9, 53.5, 53.3, 53.1, 51.4, 43.6, 40.3, 39.6, 36.3, 33.3, 33.1, 31.6,28.7, 27.3, 26.9, 26.1, 25.8, 25.5, 21.0, 18.0, 16.9, 11.4. MS [ESI⁺]:calculated for C₄₁H₆₁N₁₁O₁₀S: 899.43, observed: 900.9 [M+H]⁺. Calculatedanalysis for C₄₁H₆₁N₁₁O₁₀S: C, 54.71; H, 6.83; N, 17.12; observed C,54.73, H, 6.82, N, 17.11.

Characterization of Compound 25

Yield: 60%. (White solid). [α]_(D) ²²=−65.83 (c=1.15, CHCl₃). ¹H NMR(400 MHz, Acetone-D6): δ 1.27 (m, 1H, H-5), 1.30 (s, 6H, C(CH₃)₂ Pmc),1.45 (s, 9H, C(CH₃)₃), 1.52 (m, 1H, Hβ Arg), 1.59 (m, 1H, H-7), 1.60 (m,1H, Hy Arg), 1.62 (m, 1H, Hβ Arg), 1.81 (m, 2H, CH₂CH₂Ar Pmc), 1.98 (m,1H, Hy Arg), 2.0 (m, 1H, H-8), 2.1 (s, 3H, CH₃ Pmc), 2.38 (m, 1H, H-5),2.42 (m, 1H, H-7), 2.43 (m, 1H, H-8), 2.56 (s, 3H, CH₃ Pmc), 2.58 (s,3H, CH₃ Pmc), 2.6 (m, 1H, Hβ Asp), 2.62 (m, 2H, CH₂CH₂Ar Pmc), 2.9 (m,1H, H-4), 2.95 (m, 1H, Hβ Asp), 3.19 (m, 1H, Hδ Arg), 3.25 (m, 1H, HδArg), 3.37 (m, 2H, CH₂N3), 3.62 (d, 1H, J=13.3 Hz, Hα Gly), 4.1 (m, 1H,H-6), 4.12 (m, 1H, Hα Gly), 4.25 (m, 2H, H-9), 4.39 (m, 1H, H-3), 4.6(m, 1H, Hα Arg), 4.67 (m, 1H, Hα Asp), 6.12 (bs, 1H, (NH)₂C═NH), 6.35(bs, 2H, (NH)₂C═NH), 7.32-7.48 (m, 2H, NH Arg, NH bicyclic), 7.82 (bs,1H, NH Gly), 8.1 (bs, 1H, NH Asp). ¹³C NMR HETCOR (400 MHz, Acetone-D6):δ 62.5, 55.7, 53.4, 51.2, 42.7, 40.6, 40.3, 36.3, 35.5, 34.9, 33.4,33.3, 30.0, 28.4, 28.3, 27.5, 26.0, 22.1, 20.8, 17.9, 16.7, 13.2, 11.4.MS [FAB⁺]: calculated for C₄₀H₅₉N₁₁O₁₀S: 885.42, observed: 886 [M+H]⁺.Calculated analysis for C₄₀H₅₉N₁₁O₁₀S: C, 54.22; H, 6.71; N, 17.39;observed C, 54.21; H, 6.73; N, 17.38.

Characterization of Compound 27

Yield: 75%. (White solid). [α]_(D) ²²=−35.74 (c=1.2, CHCl₃). ¹H-NMR (400MHz, CDCl₃): δ 1.32 (s, 6H, C(CH₃)₂ Pmc), 1.46 (s, 9H, C(CH₃)₃), 1.48(m, 1H, H-6), 1.5 (m, 1H, Hβ Arg), 1.60 (m, 1H, Hy Arg), 1.62 (m, 1H, HβArg), 1.65 (m, 1H, H-5), 1.7 (m, 1H, H-4), 1.73 (m, 1H, H-8), 1.82 (m,2H, CH₂CH₂Ar Pmc), 1.96 (m, 1H, Hy Arg), 2.02 (m, 1H, H-9), 2.1 (m, 1H,H-5), 2.11 (s, 3H, CH₃ Pmc), 2.21 (m, 1H, H-8), 2.23 (m, 1H, H-9), 2.45(m, 1H, HP Asp), 2.56 (s, 3H, CH₃ Pmc), 2.58 (s, 3H, CH₃ Pmc), 2.64 (m,2H, CH₂CH₂Ar Pmc), 2.87 (m, 1H, Hβ Asp), 3.18 (m, 2H, Hδ Arg), 3.22 (m,1H, HCHN₃), 3.53 (d, 1H, J=13.0 Hz, Hα Gly), 3.62 (m, 1H, HCHN₃), 4.24(m, 1H, H-7), 4.26 (m, 1H, Hα Gly), 4.41 (m, 2H, H-10), 4.59 (m, 1H, HαArg), 4.61 (m, 1H, H-3), 4.96 (m, 1H, Hα Asp), 6.32 (bs, 3H, (NH)₂C═NH),7.46-7.58 (m, 3H, NH Gly, NH Arg, NH bicyclic), 7.9 (bs, 1H, NH Asp).¹³C NMR HETCOR (400 MHz, CDCl₃): δ 63.4, 58.9, 55.0, 53.8, 51.9, 49.7,44.3, 40.5, 39.3, 35.2, 33.0, 32.5, 32.6, 31.2, 28.0, 27.8, 26.7, 25.3,21.3, 18.6, 17.3, 11.9. MS [FAB⁺]: calculated for C₄₁H₆₁N₁₁O₁₀S: 899.43,observed: 901 [M+H]⁺. Calculated analysis for C₄₁H₆₁N₁₁O₁₀S: C, 54.71;H, 6.83; N, 17.12; observed C, 54.70; H, 6.83; N, 17.11.

b) Hydrogenation of the Azide Group

To a solution of product 21, 23, 25 or 27 (0.03 mmol) in MeOH (1 ml) isadded a catalytic amount of 10% Pd/C. The suspension is kept stirringunder hydrogen atmosphere for 4 hours. After this period of time thereaction mixture is filtered over a bed of Celite, the organic phase isevaporated to dryness and crude amines are used in the followingreaction without any further purification.

c) Synthesis of the Amides 22, 24, 26 and 28

To a solution of R₆COOH (0.043 mmol) and HBTU (0.050 mmol) in anhydrousCH₂Cl₂ (0.5 mL), a solution of 21-NH₂ (or 23-NH₂ or 25-NH₂ or 27-NH₂)(0.023 mmol) and DIPEA (0.115 mmol) in anhydrous CH₂Cl₂ (0.5 mL) wasadded dropwise. The reaction mixture was stirred at room temperature for18 h then a further amount of CH₂Cl₂ (3 mL) was added. The solution waswashed with saturated aqueous NaHCO₃ (3×5 mL) and then with 1M aqueousKHSO₄ (1×5 mL). The organic phase was separated, dried (Na₂SO₄) andevaporated. The residue was purified by flash-chromatography. The solidobtained was dissolved in TFA/thioanisole/1,2-ethandithiol/anisole(90:5:3:2, v/v/v/v; 1 mL) and the solution was stirred for 4 h at roomtemperature. The mixture was evaporated, the residue dissolved in H₂O (4mL) and the solution washed with diisopropyl ether (2×5 mL). The aqueousphase was evaporated and the residue purified by preparative HPLC(eluent A: 97% H₂O, 3% CH₃CN+0.1% TFA; eluent B: CH₃CN+0.1% TFA; flow: 4mL/min; stationary phase: Waters X-terra RP18 column 5 μm, 19×50 mm;detection: UV, k: 220 nm) to obtain product 22 (or 24 or 26 or 28) aswhite solid.

Preparation of Chelating Ligands

Preparation of the compound 5 of FIG. 8A, hereinafter referred to ascompound F.

a) Preparation of the Intermediate B

2-Allyloxyethanol A (20.2 mL; 19.2 g; 189 mmol) was dissolved inanhydrous THF (115 mL) and Na (4.12 g; 1.18 mmol) was added in littleportions at 0° C. The mixture was stirred at room temperature for 2 hthen anhydrous THF (22 mL) was added affording the precipitation of awhite solid. The suspension was stirred at room temperature for 1 day.Bromoacetic acid (12.4 g; 0.09 mmol) dissolved in anhydrous THF (20 mL)was added dropwise and the solution was stirred for 48 h. The mixturewas diluted with EtOH (150 mL) and then water (20 mL) keeping thetemperature at is 0° C. The solvents were evaporated under vacuum toobtain a yellow oil. The residue was dissolved in water (150 mL) and thesolution washed with Et₂O (2×100 mL) and CHCl₃ (2×100 mL). 37% HCl wasadded to the aqueous phase to reach pH 1 and the solution was extractedwith CHCl₃ (4×100 mL). The organic layer was separated, dried (Na₂SO₄)and evaporated under vacuum to obtain compound B (5.42 g; 71.8 mmol).Yield 38%.

MS (ESI⁺): C₇H₁₂O₄; calc. 160.07. found 161.1 (M+H)⁺.

b) Preparation of the Intermediate C

A solution of benzyl bromide (4.56 mL; 6.57 g; 38.0 mmol) in toluene (30mL) was added dropwise to a solution of compound B (5.13 g; 32.0 mmol)and DBU (4.77 mL; 4.87 g; 32.0 mmol) in toluene (70 mL). After 2 h themixture was filtered and evaporated under vacuum. The residue wasdissolved in CHCl₃ (50 mL) and the solution washed with water (3×50 mL).The organic layer was separated, dried (Na₂SO₄) and evaporated undervacuum. The residue (7.23 g) was purified by flash-chromatography(eluent: 80:20 v/v petroleum ether/EtOAc) to obtain compound C (5.40 g;21.6 mmol) as a yellow oil. Yield 68%.

MS (ESI⁺): C₁₄H₁₈O₄; calc. 250.12. found 251.1 (M+H)⁺.

c) Preparation of the Intermediate D

A solution of MCPBA (3.96 g; 23.0 mmol) in CHCl₃ (70 mL) was addeddropwise into a solution of compound C (5.20 g; 21.0 mmol) in CHCl₃ (50mL). The mixture was stirred for 48 h then washed with 10% aq. Na₂SO₃(3×150 mL) and water (3×150 mL).

The organic layer was separated, dried (Na₂SO₄) and evaporated undervacuum. The residue (4.88 g) was purified by flash-chromatography(eluent: 1:1 v/v CH₂Cl₂/Et₂O) to obtain compound D (4.05 g; 15.1 mmol)as a yellow oil. Yield 72%

MS (ESI⁺): C₁₄H₁₈O₅; calc. 266.12. found 267.1 (M+H)⁺

d) Preparation of the Intermediate E

A solution of compound D (2.44 g; 9.20 mmol) in MeCN (30 mL) was addeddropwise into a solution of DO3A tris-tert-butylester (3.19 g; 6.20mmol) and triethylamine (1.27 mL; 9.20 mmol) in MeCN (30 mL). Themixture was stirred at 50° for 32 h then cooled to room temperature andevaporated under vacuum. The residue was dissolved in CHCl₃ (50 mL) andthe solution washed with water (50 mL) and brine (50 mL). The organiclayer was separated, dried (Na₂SO₄) and evaporated under vacuum. Theresidue (6.48 g) was purified by flash-chromatography (eluent 9:1:0.1v/v/v CHCl/MeOH/NH₄OH) to obtain compound E (2.87 g; 3.66 mmol) as ayellow oil. Yield 59%.

MS (ESI⁺: C₄₀H₆₈N₄O₁₁; calc. 780.49. found 803.5 (M+Na)⁺

e) Deprotection of E to Give the Product F

10% Pd/C (28 mg) was added to a solution of intermediate E (140 mg; 0.18mmol) in MeOH (10 mL). The reaction mixture was stirred under anhydrogen atmosphere for 2 h then filtered through Millipore® apparatus(FH 0.5 μm) and evaporated to give the compound F (110 mg; 0.16 mmol) asa white oil. Yield 88%.

MS (ESI⁺: C₃₃H₆₂N₄O₁₁; calc. 690.44. found 713.4 (M+Na)⁺

Linkers

Preferred molecules useful as linkers according to the present inventionand listed in Table 1 are well known, and already marketed or they maybe easily prepared according to cited literature and/or known syntheticprocedures.

The preparation of the compound A32 of table 1 is included below as nonlimiting examples.

The starting compound of formula

was prepared according to Arosio, D. et al., Org. Biomol. Chem. 2004, 2,2113-2124. The amino group of this compound was protected with CbzCl.

The NBoc derivative was coupled with propargylamine and the Bocprotecting group was removed to give the desired compound according toscheme 7 below

Preparation of Compounds According to Formula (III) a) BiotinilatedDerivatives and Fluorescent Derivatives

The preparation of the biotinilated and fluorescent derivativescomprises the following steps:

1) Preparation of the azide derivatives of the peptidomimetic compound.

This preparation has been performed as above described, and schematizedin scheme 6 below.

2) Preparation of the linker (A32);3) coupling of the linker with the biotin and fluoresceine residues

The coupling reactions have been performed according to the followingscheme 7:

The linker, compound 17 of the scheme, was conjugated to biotin molecule19 using HBTU and DIPEA. The conjugation with fluorescein, to give thedesired conjugated compound 20, was performed adding commerciallyavailable 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester 18 to abasic solution of 17.

4) coupling reaction between the intermediate 6,8 and 20, 21 to providesthe compounds 22, 23, 24 and 25.

This step has been performed as per the following scheme 8:

The coupling reaction between the azide group on the peptidic moiety 6and 8 and the and a terminal acetilenic group on the compounds 20 and 21has been performed according to the so called “click chemistry”1,3-dipolar cycloaddition reaction, (see, f.e., Kolb, H et al, Angew.Chem. Int. Ed. 2001, 40, 2004-20021). In the present case, inparticular, the Sharpless modified Huisgen's [2+3]cycloaddition of azideand acetylene to give 1,2,3-triazoles was adopted (see, e.g.,Rostovtsev, V. V. et al. Angew. Chem. Int. Ed. 2002, 40, 2004-20021).This reaction is chemo- and regioselective, it is performed in mildconditions and, generally, is characterized by high yields.

The conjugation compounds 22, and 24 respectively have been obtained in50%-80% yield.

The same reaction performed between 6 or (and the fluoresceine tag 20,gave the final compounds 23 and 25 in higher yields (91%-92%)

b) Sugar Derivatives

The preparation has been performed according to the procedureschematized below

The compounds 11, 12, 13, and 14 have been prepared by 1,3-dipolarcycloaddition between pseudopeptides 6 and 8 and 1-O-propargyl2,3,4,6-tetra-O-acetyl-β-D-glucose 9 or 1-O-propargyl-β-D-glucose 10.The cycloaddition was performed using Cu(OAc)₂ and Na ascorbate ascatalyst in tBuOH/H2O 1:1 as per scheme 9. The reaction proceededovernight at room temperature and the desired products were isolated,after purification by falsh chromatography in good yields.

c) Chelated Complex Derivatives Preparation of the Chelated Complex 1

The preparation has been performed according to the procedureschematized below in Scheme 10.

a) preparation of the Intermedi*ate C

To a solution of compound B (0.043 mmol) and HBTU (0.050 mmol) inanhydrous CH₂Cl₂ (0.5 mL), a solution of A (0.023 mmol) and DIPEA (0.115mmol) in anhydrous CH₂Cl₂ (0.5 mL) was added dropwise. The reactionmixture was stirred at room temperature for 18 h then a further amountof CH₂Cl₂ (3 mL) was added. The solution was washed with saturatedaqueous NaHCO₃ (3×5 mL) and then with 1M aqueous KHSO4 (1×5 mL). Theorganic phase was separated, dried (Na₂SO₄) and evaporated. The residuewas purified by flash-chromatography (eluent: 9:1 CH₂Cl₂/MeOH, v/v;stationary phase: SiO₂) affording product C (0.013 mmol) as a whitesolid. Yield 57%.

MS [ESI⁺] for C₇₇H₁₂₆N₁₂O₂₁S: calc. 1586.89. found 794.7 [M+2H]²⁺.

b) Preparation of the Intermediate D

A solution of product C (0.013 mmol) inTFA/thioanisole/1,2-ethandithiol/anisole (90:5:3:2, v/v/v/v; 1 mL) wasstirred for 4 h at room temperature. The mixture was evaporated, theresidue dissolved in H₂O (4 mL) and the solution washed with diisopropylether (2×5 mL). The aqueous phase was evaporated and the residuepurified by preparative HPLC (eluent A: 97% H₂O, 3% CH₃CN+0.1% TFA;eluent B: CH₃CN+0.1% TFA; flow: 4 mL/min; stationary phase: WatersX-terra RP18 column 5 μm, 19×50 mm; detection: UV, λ: 220 nm) to obtainproduct D (0.012 mmol) as a white solid. Yield 93%.

MS [ESI⁺] for C₃₉H₆₀N₁₂O₁₈: calc. 984.41. found 985.4 [M+H]H⁺, 493.2[M+2H]²⁺.

c) Preparation of the Intermediate E

To a solution of compound D (0.010 mmol) in H₂O (5 mL) a 6.20 mM aqueoussolution of GdCl (1.77 mL) was slowly added adjusting the pH to 7 with0.05 M aqueous NaOH (0.605 mL). The mixture was loaded onto a XAD 16.00column (5 mL) and the desired compound was eluted with a MeCN/H₂Ogradient. The fractions containing the product were evaporated to givecompound E (0.009 mmol) as a white solid. Yield 90%.

MS [ESI^(+]) for C₃₉H₅₇GdN₁₂O₁₈: calc. 1139.32. found 569.7 [M+2H]².

Preparation of the Chelated Complex 2

The preparation has been performed according to the procedureschematized below in Scheme 11.

Preparation of the Product G

The same procedure described in Scheme 10 was applied to compounds A andF to obtain product G.

Chelated Complexes

Generally speaking, the paramagnetic complexes of the invention and,particularly, the Gd(III) chelated may be prepared by stoichiometricaddition of suitable Gd(III) derivatives, particularly Gd(III) salts oroxides.

Preferably, Gd(III) chloride or oxide is employed by working accordingto well known experimental methods, for instance as reported in EP230893.

d) Lipid Derivatives

The preparation of the peptidomimetic derivative I including aN-Succinyl-dioctadecylamine moiety as lipophilic unit was performedaccording to the procedure schematized below in Scheme 12.

In particular, the same procedure described in Scheme 10 was applied tocompounds A and H to obtain product I.

Multimeric Aggregate of the Invention LIPOCEST as MR Imaging Reporters

With LIPOCEST as MR Imaging detectable moiety, as used herein, we intenda paramagnetic liposomes that act as CEST agents (LIPOCEST agents) foruse in CEST imaging protocols.

LIPOCEST agents according to the invention can be prepared following anyestablished protocols for liposome preparation.

The most used procedure for preparing large unilamellar liposomes (LUV)is the “thin film hydration method”.

Briefly, a mixture of the lipidic components of the liposome membrane(phospholipids, cholesterol, amphiphilic paramagnetic complex at a givenmolar ratio) are dissolved in an organic solvent (usually achloroform/methanol mixture). The solvent is slowly evaporated in orderto obtain a thin lipidic film which is further dried under vacuum forca. 2 h. The film is hydrated, at a given temperature (usually ca. 55°C.) and under vortexing, with an aqueous solutions which may contain thehydrophilic paramagnetic metal complex to be encapsulated. This solutioncan be ipo-, iso, or hyper-tonic. The resulting suspension containingmultilamellar vesiscles (MLV) is extruded several times (usually ≧5)through polycarbonate filters with well-defined pore size (50 to 200nm). After the extrusion, the paramagnetic metal complex which has notencapsulated is exhaustively removed by dialysis against an isotonicbuffer. Besides the typical liposome characterisation (determination ofsize, polydispersity, Zeta potential, and the like), LIPOCEST agentshave to be studied in order to determine: i) the chemical shiftdifference between the resonances of intraliposomal water and of that ofthe extraliposomal water proton, and ii) their ST efficiency.

Δ^(LIPO) values can be simply determined by recording a ¹H-NMR spectrumof the LIPOCEST suspension. Alternatively, the same information can begained by collecting a Z-spectrum, in which the intensity of the waterproton signal is measured as a function of the irradiation frequency.Besides the

^(LIPO) determination, the Z-spectrum is very useful for assessing thesaturation transfer efficiency of the LIPOCEST agent. This is usuallydone by plotting the data obtained in the Z-spectrum in the ST % form.

According to the osmolarity of the aqueous solution used for hydratingthe lipidic film, LIPOCEST agents can be classified in two main groups:

i) standard LIPOCEST (S-LIPOCEST), if the aqueous solution is iso- orhyper-tonic. The paramagnetic metal complex can be only encapsulated(S-LIPOCEST-E), only incorporated in the membrane (S-LIPOCEST-I), orboth (S-LIPOCEST-EI).ii) not-standard LIPOCEST (NS-LIPOCEST), if the aqueous solution isipotonic.

The paramagnetic metal complex can be only encapsulated (NS-LIPOCEST-E),only incorporated (NS-LIPOCEST-I), or both (NS-LIPOCEST-EI)

The most important difference between S- and NS-LIPOCEST agents is thatthe latter display larger Δ^(LIPO) values.

Analogously to liposomes, LIPOCEST agents can be sterically protected inorder to increase their in vivo stability. Usually, this is done byincorporating PEGylated phospholipids in the membrane.

Targeting LIPOCEST agents can be prepared by using two main approaches:

-   -   according to the first one, the recognition vector is        incorporated in the liposome membrane and the LIPOCEST agent can        interact directly to the biological target.    -   according to the second one the LIPOCEST-target recognition is        indirect and the two units can interact through the presence of        a third component which promotes the recognition. For instance        if both the vector and the LIPOCEST are biotinylated, the        targeting protocol requires the presence of avidin or        streptavidin. The biotinylation of the LIPOCEST can be        successfully carried out by incorporating in the membrane        biotinylated phospholipids.

The following examples may best disclose this aspect of the invention.

Example 1 Preparation and characterisation of biotinylated LIPOCESTagents encapsulating [Tm-20]⁻ Preparation

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC), Cholesterol,and1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000](Ammonium Salt) (DSPE-PEG2000Biotin) were dissolved in achloroform/methanol 3:1 mixture. The molar ratio of the components(POPC/Chol/DSPE-PEG2000-Biotin) was 55:40:5 and the total amount oflipids was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Tm-20]⁻0.2 M.(chelator 20 structure is shown in of FIG. 8C) The suspension wasvortexed at 55° C. and then extruded (55° C., 4000 kPa) 5 times throughpolycarbonate filters (diameter 200 nm).

The resulting biotinylated liposomes were dialysed in order to removethe not-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The mean size of this LIPOCEST preparation was 250 nm (PDI=0.08).

FIG. 10 reports the ¹H-NMR spectrum (14.1 T, 298 K) of this preparation.The chemical shift difference between intraliposomal and bulk water was3 ppm.

FIG. 11 reports the normalised Z-spectrum of this preparation (7 T,irrad. pulse: rectangular, irrad. power 6 mT, irrad. time 2 s, 312 K).The occurrence of the saturation transfer is evident by looking at thehump visible in the downfield side of the spectrum.

FIG. 12 reports the corresponding ST-spectrum of this preparation. A ST% of ca. 70% was observed at 3 ppm from bulk water.

Example 2 Preparation and characterisation of biotinylated LIPOCESTagents incorporating Tm-21a and encapsulating [Tm-20]⁻

Preparation 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC),Cholesterol,1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-Biotinyl(PolyethyleneGlycol)2000](Ammonium Salt) (DSPE-PEG2000Biotin), and Tm-21a complex(chelator 21a is shown in FIG. 8C) were dissolved in achloroform/methanol 3:1 mixture. The molar ratio of the components(POPC/Chol/DSPE-PEG2000-Biotin/Tm-21a) was 43:28:4:25 and the totalamount of the components was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Tm-20]⁻0.02 M. Thesuspension was vortexed at 55° C. and then extruded (55° C., 4000 kPa) 5times through polycarbonate filters (diameter 200 nm).

The resulting biotinylated liposomes were dialysed in order to removethe not-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The Mean Size of this LIPOCEST Preparation was 170 nm (PDI=0.1)

FIG. 13 reports the ¹H-NMR spectrum (14.1 T, 298 K) of this preparation.The chemical shift difference between intraliposomal and bulk water was15 ppm. FIG. 14 reports the normalised Z-spectra of diluited samples ofthis preparation (7 T, irrad. pulse: rectangular, irrad. power 6 mT,irrad. time 2 s, 312 K). A ST % of ca. 10% was still detected in thesample diluited 64 times.

Example 3

Preparation and characterisation of biotinylated LIPOCEST agentsencapsulating [Tm-21](chelator 21 is shown in FIG. 8C).

Preparation

1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000](Ammonium Salt) (DSPE-PEG2000Biotin) were dissolved in achloroform/methanol 3:1 mixture. The molar ratio of the components(DPPC/DSPE-PEG2000-Biotin) was 95:5 and the total amount of thecomponents was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Tm-21]0.04 M. Thesuspension was vortexed at 55° C. and then extruded (55° C., 4000 kPa) 5times through polycarbonate filters (diameter 200 nm).

The resulting biotinylated liposomes were dialysed in order to removethe not-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The mean size of this LIPOCEST preparation was 160 nm (PDI=0.1) FIG. 15reports the ¹H-NMR spectrum (14.1 T, 298 K) of this preparation. Thechemical shift difference between intraliposomal and bulk water was 10ppm.

Example 4

Preparation and characterisation of biotinylated LIPOCEST agentsincorporating Gd-21a and encapsulating [Gd-21].

Preparation

1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC),1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000](Ammonium Salt) (DSPE-PEG2000Biotin), and Gd-21a complexwere dissolved in a chloroform/methanol 3:1 mixture. The molar ratio ofthe components (DPPC/DSPE-PEG2000-Biotin/Gd-21a) was 60:5:35 and thetotal amount of the components was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Gd-21]0.04 M. Thesuspension was vortexed at 55° C. and then extruded (55° C., 4000 kPa) 5times through polycarbonate filters (diameter 200 nm).

The resulting biotinylated liposomes were dialysed in order to removethe not-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The mean size of this LIPOCEST preparation was 140 nm (PDI=0.1) FIG. 16reports the ¹H-NMR spectrum (14.1 T, 298 K) of this preparation. Thechemical shift difference between intraliposomal and bulk water was 19.3ppm.

Example 5 Preparation and Characterisation of biotinylated LIPOCESTAgents Incorporating Dy-21a and Encapsulating [Tm-20]⁻ Preparation

1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC),1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000](Ammonium Salt) (DSPE-PEG2000Biotin), and Dy-21a complexwere dissolved in a chloroform/methanol 3:1 mixture. The molar ratio ofthe components (DPPC/DSPE-PEG2000-Biotin/Gd-21a) was 60:5:35 and thetotal amount of the components was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Tm-20]⁻0.02 M. Thesuspension was vortexed at 55° C. and then extruded (55° C., 4000 kPa) 5times through polycarbonate filters (diameter 200 nm).

The resulting biotinylated liposomes were dialysed in order to removethe not-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The mean size of this LIPOCEST preparation was 160 nm (PDI=0.1) FIG. 17reports the ¹H-NMR spectrum (14.1 T, 298 K) of this preparation. Thechemical shift difference between intraliposomal and bulk water was −36ppm.

Example 6

Preparation and characterisation of LIPOCEST agents encapsulating[Tm-20]⁻ and incorporating a compound of formula (III) wherein R₆ is theN-Succinyl-dioctadecylamine lipid moiety.

Preparation

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC), Cholesterol,1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[methoxy(Polyethyleneglycol)2000](Ammonium Salt) (DSPE-PEG2000), and the peptidomimeticderivatives according to formula (III) comprisingN-Succinyl-dioctadecylamine as lipid moiety, (lipid derivative I ofscheme 12) were dissolved in a chloroform/methanol 3:1 mixture. Themolar ratio of the components (POPC/Chol/DSPE-PEG2000/targeting lipid)was 55:40:2.5:2.5 and the total amount of the components was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Tm-20]⁻0.2 M. Thesuspension was vortexed at 55° C. and then extruded (55° C., 4000 kPa) 5times through polycarbonate filters (diameter 200 nm).

The resulting liposomes were dialysed in order to remove thenot-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The mean size of this LIPOCEST preparation was 200 nm (PDI=0.1)

FIG. 18 reports the ¹H-NMR spectrum (14.1 T, 298 K) of this preparation.The chemical shift difference between intraliposomal and bulk water was3 ppm.

Example 7 Visualization of Integrin Receptors by Use of a PeptidomimeticDerivative According to Formula (III) wherein R₆ is a Biotin Moiety anda LIPOCEST Agent

Preparation and characterisation of and cell targeting of biotinylatedLIPOCEST agents incorporating Tm-21a and encapsulating [Tm-20]⁻.

Preparation

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC), Cholesterol,and1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000](Ammonium Salt) (DSPE-PEG2000Biotin), and Tm-21a complexwere dissolved in a chloroform/methanol 3:1 mixture. The molar ratio ofthe components (POPC/Chol/DSPE-PEG2000-Biotin/Tm-21a) was 43:28:4:25 andthe total amount of lipids was 20 mg.

The solution was slowly evaporated in order to obtain a thin lipidicfilm which was further dried under vacuum for 2 hours.

The film was hydrated with 1 mL of a solution of [Tm-20]⁻ 0.02 M. Thesuspension was vortexed at 55° C. and then extruded (55° C., 4000 kPa) 5times through polycarbonate filters (diameter 200 nm).

The resulting biotinylated liposomes were dialysed in order to removethe not-encapsulated metal complex (two dialysis cycles of 4 hours eachagainst an isotonic buffer at pH 7).

Characterisation

The mean size of this LIPOCEST preparation was 190 nm (PDI=0.1)

The chemical shift difference between intraliposomal and bulk water was11.0 ppm (298 K).

Cell Targeting Experiment

Ca. 1×10⁶ HUVEC (Human Umbilical Vein Endothelial Cells; see, asreference, Garlanda C. Parravicini C. Sironi M. De Rossi M. Wainstok deCalmanovici R. Carozzi F. Bussolino F. Calotta F. Mantovani A. VecchiA., Progressive growth in immunodeficient mice and host cell recruitmentby mouse endothelial cells transformed by polyoma middle-sized Tantigen: implications for the phatogenesis of opportunistic vasculartumors; 1994, Proc Natl Acad Sci USA, 91, 7291-7295) cells were detachedfrom the cell culture medium by EDTA (0.3 g/L) and washed three timeswith ice-cold PBS buffer. About 5×10⁵ cells were suspended in 200 μL ofa solution containing the biotinylated targeting peptide of theinvention, i.e. a peptidomimetic derivative according to formula (III)wherein R₆ is a biotin moiety, (60 μM) and incubated at 4° C. After 15mins of incubation, the cells were washed two times with ice-cold PBSbuffer and suspended in 200 μL of a solution containing streptavidin (2μM). After 15 mins of incubation at 4° C., the cells were washed twotimes with ice-cold PBS buffer and suspended in 200 L of a suspensioncontaining the biotinylated LIPOCEST. Finally, after 15 mins ofincubation at 4° C., the cells were washed three times with ice-cold PBSbuffer and the cellular pellet was imaged by MRI.

In parallel, the remaining HUVEC cells (ca. 5×10⁵) were used as controland were only washed three times with ice-cold PBS buffer.

FIG. 19 reports the ST spectra of the two cellular pellets of Example 7,including, the first HUVEC cells treated with the biotinilated LIPOCESTand the second only HUVEC cells as control (7 T, irrad. pulse:rectangular, irrad. power 12 PT, irrad. time 2 s, 293 K). The treatedHUVEC cells displays a maximum Saturation Transfer % of ca. 4% uponirradiation at 10 ppm from the resonance of bulk water.

FIG. 20 reports the corresponding CEST-MR images [on-(irradiation at 10ppm from bulk water), off-(irradiation at −10 ppm from bulk water), and(on-off)] of the phantom made of two capillaries containing the twocellular pellets. Importantly, only the treated HUVEC cells are visiblein the on-off difference image when the irradiation is carried out at 10ppm from the bulk water.

Pharmacokinetic Determination Protocol Outline Introduction

Biological tests for the evaluation of the affinity of the compounds ofthe invention towards the αvβ3 and αvβ5 integrins may be performed usingknown tests also described in the literature [C. C. Kumar, H. Nie, C. P.Rogers, M. Malkowski, E. Maxwell, J. J. Catino, L. Armstrong, J.Pharmacol. Exp. Ther. 1997, 283, 843] for example such as the onereported in patent application EP 1 077 218.

In the present case, the following protocol has been applied.

Materials

Test articles

Reagents

Compound: Penicillin/Streptomycin (10000 μg/mL)

Supplier: Euroclone®, Wetherby, West York, UK

Compound: L-Glutamine (200 mM)

Supplier: Biochrom KG, Berlin, Germany

Compound: Foetal Calf Serum (FCS)

Supplier: HyClone®, Logan, Utah, U.S.A.

Compound: Dulbecco's Modified Eagle's Medium (DMEM)

Supplier: SIGMA Chemicals, St. Louis, Mo., U.S.A.

Compound: Non-essential amino acids

Supplier: SIGMA Chemical Co, Sigma-Aldrich. Chem. GMBH Berlin, Germany

Compound: Sodium pyruvate

Supplier: SIGMA Chemical Co, Sigma-Aldrich. Chem. GMBH Berlin, Germany

Compound: Dulbecco's Phosphate Buffered Saline (PBS-)

Supplier: SIGMA Chemical Co, Sigma-Aldrich. Chem. GMBH Berlin, Germany

Compound: Trypsin-EDTA

Supplier: SIGMA Chemical Co, Sigma-Aldrich. Chem. GMBH Berlin, Germany

Compound: 0.9% Sodium Chloride solution

Supplier: S.A.L.F. SpA Laboratorio Farmaceutico, Bergamo, Italy

Compound: Endothelial Cell Basal Medium-2 (EBM-2)

Supplier: Cambrex Bio Science Walkersville Inc., Walkersville, Md.,U.S.A.

Compound: EGM-2 MV Single Quots

Supplier: Cambrex Bio Science Walkersville, Inc. Walkersville, Md.,U.S.A.

Mouse anti-human integrin

v

3 monoclonal antibody

Supplier: Chemicon International Inc., 28835 Single Oak Drive, TameculaCalif.

Goat anti-mouse IgG fluorescein conjugated antibody

Supplier: Chemicon International Inc., 28835 Single Oak Drive, TameculaCalif.

Vitronectin from Human Plasma

Supplier: SIGMA Chemical Co, Sigma-Aldrich. Chem. GMBH Berlin, Germany

Cycle(Arg-Gly-Asp-D-Phen-Val) peptide

Supplier: Bachem AG, 4416 Bubendorf, Switzerland

Human Integrin

v

3 purified protein

Supplier: Chemicon International Inc., 28835 Single Oak Drive, TameculaCalif.

NeutrAvidin horseradish peroxidase conjugated

Supplier: Pierce Biotechnology Inc., Rockford, Ill. 61105

Test System

Justification of the choice of the test system: Endothelial cells fromdifferent origins and tumor cell lines expressing αvβ₃ or αvβ₅ receptorshave been chosen as test system since they are suitable to test thespecific binding of cyclic peptides to αvβ₃ integrin receptor in vitro.

Biological Samples

H5V cell lines were grown in 90% DMEM supplemented with 2 mML-glutamine, 100 μg/mL Penicillin/Streptomycin, 0.1 mM non-essentialamino acids, 1.0 mM sodium pyruvate and 10% FCS.

HUVEC cells were grown in Endothelial Cell Basal Medium-2 supplementedwith EGM-2 MV Single Quots.

Location of Experimental Phases Methods Experimental Procedures FACSAnalysis

Selected cell lines were detached at confluence by treatment with 0.02%EDTA, washed in PBS and maintained in suspension for 1 hour in culturemedium. Cells were then incubated with the primary antibody anti-humanintegrin αvβ₃ for 1 hour at 4° C., washed in PBS and incubated with aspecific FITC-labelled secondary antibody at the same conditions. Afterwashing in PBS, cell surface fluorescence were analyzed by flowcytometer fuorescence-activated cell sorting (FACS) scan.

The same procedure is utilized to stain the cells with FITC-labelledintegrin antagonists.

Adhesion Assay

96-wells tissue culture plates were coated with either fibronectin orvitronectin (5 μg/mL), overnight at 4° C. Selected cell lines wereseeded in each well and allow to adhere for 1 or 3 hours at 37° C. inthe presence of various concentration of integrin antagonists or primaryantibody anti-human integrin

v

3 as positive control. Non adherent cells were removed with PBS and theremaining cells were fixed with 3.5% paraformaldehyde for 10 minutes,stained with 0.5% Crystal violet for 10 minutes and washed with water.Stained cells were solubilized and the amount of adherent cells werequantified by measuring the absorbance at 575 nm on a microtiter platereader. Experiments were done in six replicates and repeated at leasttwice. Results are expressed as mean+/−SD compound concentration thatinhibits 50% of cell adhesion.

Ligand Binding Assay

Purified integrin αvβ₃ and αvβ₅ receptors were diluted to 0.5 or 1 g/mLin coating buffer containing 20 mmol/L Tris-HCl (PH 7.4), 150 mmol/LNaCl, and 1 mmol/L MnCl₂ in the presence of 2 mmol/L CaCl₂ and 1 mmol/LMgC12. An aliquot of diluted receptors (100 μL/well) was added to96-well microtiter plates and incubated overnight at 4° C. Then theplates were washed and the aspecific binding sites were blocked withcoating buffer plus 1-2% bovine serum albumin at room temperature for 2hours. The blocking buffer was removed and the wells were washed 3 timesand incubated in quadruplicate with different concentration (0.1-10 μM)of integrin antagonists and 1 μg/mL biotinylated human vitronectin atroom temperature for 3 hours as standard competitors. The plates werewashed 3 times and the bounded competitor was detected usingstrepto-avidin-HRP conjugate at 0.01 μg/well (3).

The integrin antagonist concentration producing 50% inhibition ofvitronectin binding to purified protein was calculated.

Proliferation Assay

For the proliferation assay, selected cell lines were seeded in 96-wellplates in complete medium. After 24 hours the medium were removed andreplaced with fresh medium containing scalar concentration of integrinantagonists. The plates were incubated for additional 72 hours beforeassessing the cell proliferation by MTT or other alternativeproliferation assay.

Immunofluorescence

Selected cell lines were detached at confluence by treatment with 0.02%EDTA, washed in PBS and were seeded on a glass coverlip up toconfluence. Cells were fixed, then incubated with the FITC-labelledintegrin antagonist or with the primary antibody anti-human integrinαvβ₃ for 1 hour, washed in PBS, containing 0.1% BSA, and incubated withthe specific FITC-labelled secondary antibody. After washing in PBS, theintegrin receptor positive cells were analyzed byfluorescent-microscopy.

Platelet Aggregation Assay

The platelet aggregation response to 11-mer thrombin receptor-activatingpeptide (25-100 μmol/L) was measured in platelet-rich plasma from guineapig, human or rabbit. The platelet concentration was adjusted inplatelet-poor plasma to 3×10⁸/mL and platelet aggregation was determinedwithin 1 hour by turbidometric method in a dual-channel aggregometer.Vehicle or different concentrations of peptide was added toplatelet-rich plasma 1 minute before starting aggregation. The extent ofplatelet aggregation was quantified as the maximum change in lighttransmittance within 4 minutes after the addition of the agonist. Theresults were expressed as the antagonist concentration that inhibit 50%platelet aggregation.

In Vitro Invasion Assay

The invasion assay was performed in the transwell system. A suspensionof selected tumor cell lines (0.5−1×10⁶/mL) was added in the uppercompartment of the transwell insert coated with different basementproteins in presence of integrin receptor antagonists. The transwellswere incubated for 24 hours before to remove the non invading cells andthe matrix proteins. The migrating and invading cells on the lowersurface of transwell unit were stained and quantified by opticalmicroscope.

In Vitro Vessel Formation Assay

The vessel formation assay was performed according to the “In vitroangiogenesis assay kit” purchased from Chemicon international.

Parties to the joint research agreement are the University ofMilan—Center of Excellence, Institute of Science and MolecularTechnology—CNR (National Research Council), Colosseum CombinatorialChemistry Centre for Technology—University of Rome—Tor Vergata,Immune-Biological Research Institute of Siena (Chiron Group), BraccoImaging S.p.A., Inpeco, and Pharmacia-Italy.

REFERENCES

-   J. J. Marugan et all. Design, Synthesis and biological evaluation of    novel potent and selective αvβ₃/αvβ₅ integrin dual inhibitors with    improved bioavalability. Selection of the molecular care. J. Med.    Chem. 2005, 48, pp. 926-934.-   L. Belvisi et all. Biological and molecular properties of a new    αvβ₃/αvβ₃ integrin antagonist. Mol Cancer Ther 2005, 4(11), pp.    1670-1680.-   R. Haubner et all. Structural and functional aspects of    RGD-containing cyclic pentapeptides as highly potent and selective    integrin αvβ₃ antagonists. J. Am. Chem. Soc. 1996, 118, pp.    7461-7472.

We claim:
 1. A compound of formula (III)

wherein n is 1 or 2; p is an integer between 1 and 5; OR₄ and R₅together constitute the sequence Asp-Gly-Arg; R₆ is a biologicallyactive moiety selected from the group consisting of aradiotherapeutically or diagnostically effective molecule, aphospholipid or lipid moiety, and a biotin or an avidin residue; and Lis a group (i) —CONH—, (ii) —NHCONH—, (iii) —HCSNH—,

or is a divalent linking moiety comprising two of the groups (i), (ii),(iii), (iv), and/or (v) as terminal groups; or the compound of formula(III) is selected from the group consisting of salts, individualenantiomers, individual diastereoisomers, and racemic mixtures of thecompound of formula (III) in whatever proportion.
 2. The compound ofclaim 1 wherein L is the group (i), (ii), (iii), (iv) or (v).
 3. Thecompound of claim 1 wherein L is a divalent linking moiety comprisingtwo of the groups (i), (ii), (iii), (iv) and/or (v) as terminal groups.4. The compound of claim 3 wherein L is selected from the groupconsisting of substituted or unsubstituted, either saturated orunsaturated, straight or branched alkyl chains; peptides from straight,branched or cyclic amino acid chains composed from a single amino acidor from different amino acids; derivatized or underivatized polyethyleneglycol, polyoxyethylene, or polyvinylpyridine chains; substituted orunsubstituted polyamide chains; derivatized or underivatized polyamine,polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol),polyglycerol, or oligosaccharide chains; glycosylated amino acidresidues, alternating block copolymers; malonic, succinic, glutaric,adipic and pimelic acids; caproic acid; simple diamines and dialcohols.5. The compound of claim 4 wherein L is derived from linkers A1 to A32:LINKER A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

A21

A22

A23

A24

A25

A26

A27

A28

A29

A30

A31

A32

or from a suitable combination thereof.
 6. The compound of claim 4wherein L is a divalent linking moiety selected from the groupconsisting of —CONH—CH₂—(CH₂—O—CH₂)₄—CONH—, —CONH—(CH₂)₃—NHCONH—, and—CONH—CH(CH₃)—CONH—.
 7. The compound of claim 1 wherein R₆ is adiagnostically effective molecule comprising an imaging detectablemoiety.
 8. The compound of claim 7 wherein R₆ is a chelated orpolychelated complex of a paramagnetic metal ion selected from the groupconsisting of: Fe(²⁺⁾, Fe(³⁺⁾, Cu(²⁺⁾, Ni(²⁺), Rh(²⁺), Co(²⁺), Cr(³⁺),Gd(³⁺), Eu(³⁺), Dy(³⁺), Tb(³⁺), Pm(³⁺), Nd(³⁺), Tm(³⁺), Ce(³⁺), Y(³⁺),Ho(³⁺), Er(³⁺), La(³⁺), Yb(³⁺) Mn(³⁺), and Mn(²⁺).
 9. The compound ofclaim 8 wherein the paramagnetic metal ion is Gd(³⁺).
 10. The compoundof claim 1 wherein R₆ is a chelated or polychelated complex of atherapeutically effective radioactive metal ion that emits ionizingradiation selected from the group consisting of beta particles, alphaparticles, and Auger or Coster-Kroning electrons.
 11. The compound ofclaim 1 wherein R₆ is a chelated or polychelated complex of atherapeutically effective radionuclide selected from the groupconsisting of: ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au.
 12. The compound ofclaim 7 wherein R₆ is a chelated or polychelated complex of a gamma rayor positron emitting radionuclide.
 13. The compound of claim 12 whereinthe radionuclide is selected from the group consisting of: ⁵¹Mn, ⁵²Fe,⁶⁰Cu, ⁶⁸Ga, ⁷²As, ^(94m)Tc, ¹¹⁰In, ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc,⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹⁶⁸Yb, ¹⁴⁰La, ⁸⁸Y, ¹⁶⁵Dy, ⁶²Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru,²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁹Pd, and ¹⁹⁸Au.
 14. The compound ofclaim 8 wherein the chelator or polychelator is a compound selected fromthe group consisting of: diethylenetriamine pentaacetic acid (DTPA) andderivatives thereof;1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);1-substituted 1,4,7-tricarboxymethyl 1,4,7,10 teraazacyclododecanetriacetic acid (DO3A);1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid(DOTMA); ethylenediaminetetraacetic acid (EDTA);1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA);ethylenebis-(2-hydroxy-phenylglycine) (EHPG) and derivatives thereof;benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid; benzo-TETA, benzo-DOTMA,and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);and derivatives of 1,3-propylenediaminetetraacetic acid (PDTA),triethylenetetraaminehexaacetic acid (TTHA),1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM), and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM) or isa compound selected from the group consisting of:


15. The compound of claim 1 wherein R₆ is a phospholipid or other lipidmoiety.
 16. The compound of claim 15 wherein the phospholipid or otherlipid moiety is a compound selected from the group consisting of fattyacids, neutral fats, phosphatides, glycolipids, aliphatic alcohols,waxes, terpenes, and steroids.
 17. The compound of claim 15 wherein thephospholipid or other lipid moiety is a compound selected from the groupconsisting of phosphatidylcholines; phosphatidylethanolamines;phosphatidylserines; phosphatidylglycerols; sphingolipids; glycolipids;glucolipids; sulphatides; phosphatidic acids; palmitic fatty acids;stearic fatty acids; arachidonic fatty acids; lauric fatty acids;myristic fatty acids; lauroleic fatty acids; physeteric fatty acids;myristoleic fatty acids; palmitoleic fatty acids; petroselinic fattyacids; oleic fatty acids; isolauric fatty acids; isomyristic fattyacids; isostearic fatty acids; cholesterol and cholesterol derivatives;polyoxyethylene fatty acid esters; polyoxyethylene fatty acid alcohols;polyoxyethylene fatty acids alcohol ethers; polyoxyethylated sorbitanfatty acid esters; glycerol polyethylene glycol oxy-stearate; glycerolpolyethylene glycol ricinoleate; ethoxylated soybean sterols;ethoxylated castor oil; polyoxyethylene polyoxypropylene fatty acidpolymers; polyoxyethylene fatty acid stearates;1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoyl-glycerophosphoethanolamine;N-Succinyl-dioctadecylamine; palmitoylhomocysteine;lauryltrimethylammonium bromide; cetyltrimethyl-ammonium bromide;myristyltrimethylammonium bromide; alkyldimethylbenzylammonium chloride,wherein alkyl is a C₁₂, C₁₄, or C₁₆ alkyl; benzyldimethyldodecylammoniumbromide; benzyldimethyldodecyl ammonium chloride;benzyldimethylhexadecylammonium bromide; benzyldimethylhexadecylammoniumchloride; benzyldimethyltetradecyl ammonium bromide;benzyldimethyltetradecyl ammonium chloride; cetyldimethylethylammoniumchloride; cetylpyridinium bromide; cetylpyridinium chloride;N-[1,2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA);1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); and1,2-dioleoyl-c-(4′-trimethylammonium)-butanoyl-sn-glycerol (DOTB). 18.The compound of claim 15 wherein the phospholipid or other lipid moietyis a compound selected from the group consisting ofdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine, and diasteroylphosphatidylcholine. 19.The compound of claim 15 wherein the phospholipid or other lipid moietyis a compound selected from the group consisting ofdipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine,and N-succinil-dioleoylphosphatidyl-ethanolamine.
 20. The compound ofclaim 15 wherein the phospholipid or other lipid moiety isdipalmitoylphosphatidylserine.
 21. The compound of claim 15 wherein thephospholipid or other lipid moiety is dipalmitoyl phosphatidic acid(DPPA).
 22. The compound of claim 15 wherein the phospholipid or otherlipid moiety is selected from the group consisting of cholesterolhemisuccinate, cholesterol sulphate, andcholesteryl-(4-trimethylammonio)-butanoate.
 23. A macromolecular systemcomprising the compound as defined in claim 15 incorporated within aliposome, micelle, microemulsion, bubble, microbubble, microballoon,microsphere, paramagnetic liposome, LIPOCEST, or gas-containing vesiclefor use as a diagnostic agent.
 24. A compound selected from the groupconsisting of:


25. The compound of claim 7 wherein R₆ is selected from the groupconsisting of: a dye molecule, a fluorescent molecule, a phosphorescentmolecule, a molecule absorbing in the UV spectrum, a quantum dot, and amolecule absorbing near or far infrared radiations.
 26. The compound ofclaim 25 wherein R₆ is a fluorescent molecule.
 27. The compound of claim25 wherein R₆ is a NIR dye.
 28. The compound of claim 27 wherein the R₆is a cyanine or indocyanine derivative selected from the groupconsisting of Cy5.5, IRDye800, indocyanine green (ICG) and tetrasulfonicacid substituted indocyanine green.
 29. A multimeric construct offormula (IV)

wherein: OR₄, R₅, L, n and p are as defined in claim 1, T is ananchoring system containing at least three, optionally protected, equalor different binding sites or functional groups deriving from anypolyvalent organic residue which can be aliphatic with open chain,optionally branched, or alicyclic, or heterocyclic containing N,O,and/or S or aromatic or heteroaromatic, or it is an avidin orstreptavidin moiety; r is an integer from 2 to 10; or the multimericconstruct of formula (IV) selected from the group consisting of salts,individual enantiomers, individual diastereoisomers, and racemicmixtures of the multimeric construct of formula (IV) in whateverproportion.
 30. The multimeric construct of claim 29 wherein T isselected from the group consisting of: (a) N-branched lysine systems,(b) polycarboxylic derivatives, (c) polyaminated derivatives, and (d)amino acids.
 31. A multimeric construct of formula (VI) or formula (VII)

wherein OR₄, R₅, L n, p and R₆ are as defined in claim 1, T is selectedfrom the group consisting of N-branched lysine systems, polycarboxylicderivatives, polyaminated derivatives, and amino acids; R is an integerfrom 2 to 10; B is an integer from 2 to 5; or the multimeric constructof formula (VI) or formula (VII) selected from the group consisting ofsalts, individual enantiomers, individual diastereoisomers, and racemicmixtures of the multimeric construct of formula (VI) or formula (VII) inwhatever proportion.
 32. A multimeric construct of formula (V)

wherein OR₄, R₅, L, n and p are as defined in claim 1; R₆ is a biotinmoiety; T is a streptavidin or an avidin moiety; r is 2 or 3; or themultimeric construct of formula (V) selected from the group consistingof salts, individual enantiomers, individual diastereoisomers, andracemic mixtures of the multimeric construct of formula (V) in whateverproportion.
 33. A macromolecular aggregate comprising a liposome,micelle, microemulsion, vesicle, microsphere, paramagnetic liposome,LIPOCEST agent or gas containing vesicle having a number of biotinresidues on their surface, said biotin residues being coupled with themultimeric construct of formula (V) as defined in claim
 32. 34. Amacromolecular system comprising the multimeric construct of formula(VI) or formula (VII) as defined in claim 31 in which R₆ is aphospholipid or lipid moiety incorporated within a paramagneticliposome, LIPOCEST, or gas-containing vesicle, for use as a diagnosticagent.
 35. A compound of formula (III) as defined in claim 1 wherein R₆is a diagnostically or radiotherapeutically effective moiety for use asa diagnostic or radiotherapeutic agent, respectively.
 36. A multimericconstruct of formula (VI) or formula (VII) as defined in claim 31wherein R₆ is a diagnostically or radiotherapeutically effective moietyfor use as a diagnostic or radiotherapeutic agent, respectively.
 37. Adiagnostic or radiotherapeutic agent comprising the compound of formula(III) as defined in claim 1 wherein R₆ is a diagnostically orradiotherapeutically effective molecule.
 38. A diagnostic orradiotherapeutic agent comprising the multimeric construct of formula(VI) or formula (VII) as defined in claim 31 wherein R₆ is adiagnostically or radiotherapeutically effective molecule.
 39. Apharmaceutical composition comprising the diagnostic or radiotherapeuticagent of claim 37 and pharmaceutically acceptable excipients, carriersand/or diluents.
 40. A method of imaging a human or animal body organregion or tissue which comprises administering to said human or animalan imaging effective amount of the diagnostic agent of claim
 37. 41. Themethod according to claim 40 for in vivo imaging of a human body organ,region, or tissue affected by angiogenic process and selected fromtumorous or cancerous tissues, or inflammation, or for monitoringprogress and results of therapeutic treatments of said pathologies. 42.A method for in vitro imaging of a pathological system selected from thegroup consisting of cells, biological fluids, and tissues isolated froma live mammal patient, which method comprises contacting saidpathological system with an imaging effective amount of the diagnosticagent of claim
 37. 43. A method of treatment or prevention ofangiogenesis or a related disorder in a patient in need, which methodcomprises administering to said patient an effective amount of theradiotherapeutic agent of claim
 37. 44. A pharmaceutical compositioncomprising the diagnostic or radiotherapeutic agent of claim 37 andpharmaceutically acceptable excipients, carriers and/or diluents.
 45. Amethod of imaging a human or animal body organ region or tissue whichcomprises administering to said human or animal an imaging effectiveamount of the diagnostic agent of claim
 38. 46. The method according toclaim 45 for in vivo imaging of a human body organ, region, or tissueaffected by angiogenic process and selected from tumorous or canceroustissues, or inflammation, or for monitoring progress and results oftherapeutic treatments of said pathologies.
 47. A method for in vitroimaging of a pathological system selected from the group consisting ofcells, biological fluids, and tissues isolated from a live mammalpatient, which method comprises contacting said pathological system withan imaging effective amount of the diagnostic agent of claim
 38. 48. Amethod of treatment or prevention of angiogenesis or a related disorderin a patient in need, which method comprises administering to saidpatient an effective amount of the radiotherapeutic agent of claim 38.